Method for Treating an Individual Suffering from a Chronic Infectious Disease and Cancer

ABSTRACT

A method for treating an individual suffering from a chronic infectious disease and who has cancer employs a CRISPR system to selectively kill or reduce the numbers of pathogenic bacteria within the individual and the individual is then administered an immune checkpoint inhibitor. In particular embodiments, the pathogenic bacteria is one of E. coli, Pseudomonas aeruginosa and Klebsiella bacteria, and the checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010. Further embodiments include enhancing the growth of a second bacteria in the individual, such bacteria including Akkermansia, Bacteroides, Bifidobacterium, Clostridium, Enterococcus, Fusobacterium, Coprococcus, LactoBacillus, Propionibacterium, Ruminococcus, Veillonella, Prevotella, Escherichia and Streptococcus. The CRISPR system may include Cas9, Cpf1 and Cas3, and may be delivered using a bacteriophage.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 17/027,953, filed on Sep. 22, 2020 (now U.S. Pat. No.11,026,982, issued Jun. 8, 2021), which is a continuation-in-part ofU.S. patent application Ser. No. 16/917,096, filed Jun. 30, 2020 (nowU.S. Pat. No. 10,940,169, issued Mar. 9, 2021), which is a acontinuation-in-part of U.S. patent application Ser. No. 16/782,364,filed Feb. 5, 2020 (now U.S. Pat. No. 10,835,560, issued Nov. 17, 2020),which is a continuation-in-part of U.S. patent application Ser. No.16/423,375, filed May 28, 2019 (now U.S. Pat. No. 10,555,976, issuedFeb. 11, 2020), which is a continuation of U.S. patent application Ser.No. 16/160,336, filed Oct. 15, 2018 (now U.S. Pat. No. 10,314,866,issued Jun. 11, 2019), which is a continuation of U.S. patentapplication Ser. No. 15/403,823, filed Jan. 11, 2017 (now U.S. Pat. No.10,111,913, issued Oct. 30, 2018), which is a non-provisional of U.S.Provisional Patent Application Ser. No. 62/296,186, filed on Feb. 17,2016.

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/426,346, filed May 30, 2019 (now U.S. Pat. No. 10,716,815,issued Jul. 20, 2020), which is a continuation of U.S. patentapplication Ser. No. 15/639,767, filed Jun. 30, 2017 (now issued U.S.Pat. No. 10,314,865, issuing Jun. 11, 2019), which is acontinuation-in-part of U.S. patent application Ser. No. 15/437,976,filed Feb. 21, 2017 (now U.S. Pat. No. 9,730,967, issued Aug. 15, 2017),which is a continuation-in-part application of U.S. patent applicationSer. No. 15/228,454, filed Aug. 4, 2016 (now U.S. Pat. No. 9,585,920,issued Mar. 7, 2017), which is a continuation-in-part application ofU.S. patent application Ser. No. 14/954,074, filed on Nov. 30, 2015 (nowissued U.S. Pat. No. 9,457,077, issued Oct. 4, 2016).

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 16/776,861, filed Jan. 30, 2020, which is acontinuation of U.S. patent application Ser. No. 16/142,171, filed Sep.26, 2018 (now U.S. Pat. No. 10,548,761, issued Feb. 4, 2020), which is acontinuation-in-part of U.S. patent application Ser. No. 15/395,419,filed Dec. 30, 2016 (now U.S. Pat. No. 10,086,018, issued Oct. 2, 2018),which is a non-provisional of U.S. Provisional Patent Application Ser.No. 62/274,550, filed on Jan. 4, 2016.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 17/023,736, filed Sep. 17, 2020, which is acontinuation-in-part of U.S. patent application Ser. No. 17/011,175,filed Sep. 3, 2020, which is a continuation-in-part of U.S. patentapplication Ser. No. 16/722,117, filed Dec. 20, 2019 (now U.S. Pat. No.10,842,834, issued Nov. 24, 2020), which is a continuation-in-part ofU.S. patent application Ser. No. 16/229,252, filed Dec. 21, 2018 (nowU.S. Pat. No. 10,512,661, issued Dec. 24, 2019), which is acontinuation-in-part of U.S. patent application Ser. No. 15/392,173,filed Dec. 28, 2016 (now U.S. Pat. No. 10,245,288, issued Apr. 2, 2019),which is a non-provisional of U.S. Provisional Patent Application Ser.No. 62/275,341, filed on Jan. 6, 2016.

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/904,056, filed Jun. 17, 2020, which is acontinuation-in-part of U.S. patent application Ser. No. 15/983,250filed on May 18, 2018 (now U.S. Pat. No. 10,687,975, issued Jun. 23,2020), which is a continuation-in-part of U.S. patent application Ser.No. 15/384,716 filed on Dec. 20, 2016 (now issued U.S. Pat. No.9,987,224, issued Jun. 5, 2018), which claims priority of U.S.Provisional Patent Application Serial Nos. 62/387,405, filed on Dec. 24,2015.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 15/270,034, filed Sep. 20, 2016 (now U.S. Pat. No.9,750,802, issued Sep. 5, 2017), which is a continuation-in-partapplication of U.S. patent application Ser. No. 14/954,074, filed onNov. 30, 2015 (now issued U.S. Pat. No. 9,457,077, issuing on Oct. 4,2016).

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 16/037,053, filed Jul. 17, 2018.

The entire disclosure of the prior applications are considered to bepart of the disclosure of the accompanying application and are herebyincorporated by reference.

FIELD OF THE INVENTION

A method for treating an individual suffering from a chronic infectiousdisease and who has cancer employs a CRISPR system to selectively killor reduce the numbers of pathogenic bacteria within the individual andthe individual is then administered an immune checkpoint inhibitor. Inparticular embodiments, the pathogenic bacteria are one of E. coli,Pseudomonas aeruginosa and Klebsiella bacteria, and the checkpointinhibitor is selected from the group consisting of nivolumab,pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042,RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.Further embodiments include enhancing the growth of a second bacteria inthe individual, such bacteria including Akkermansia, Bacteroides,Bifidobacterium, Clostridium, Enterococcus, Fusobacterium,LactoBacillus, Propionibacterium, Ruminococcus, Veillonella, Prevotella,Escherichia and Streptococcus. Still other embodiments includeincreasing the levels of Roseburia and/or Faecalibacterium prausnitzii,in the individual's gut microbiome.

BACKGROUND OF THE INVENTION

There are over 200 different known cancers that afflict human beings.Cancer causes millions of deaths a year worldwide and rates are alsorising as more people live to an older age and urbanization causes morestress. It is anticipated that one in eight people currently alive willeventually die of cancer. Cancer manifests itself in a wide variety offorms, characterized by different degrees of invasiveness andaggressiveness. Malignant tumors are the second leading cause of deathin the United States, after heart disease.

The majority of tumors harbor p53 mutants. As the “guardian of thegenome,” p53 is arguably one of the most important tumor suppressorsthat controls the regulation and expression of many genes that mediatecell cycle arrest, DNA repair and apoptosis. Under physiologicalconditions, newly synthesized p53 quickly undergoes ubiquitination anddegradation. The p53 tumor suppressor protein plays critical roles inpreventing malignant transformation by inducing cell growth arrest orapoptosis. Normally, p53 is inactive in the cell and its levels are low.In response to cellular stress such as DNA damage, p53 levels increasedramatically and it becomes activated through multiple post-translationmodifications.

Cancer causes millions of deaths a year worldwide and rates are alsorising as more people live to an older age. It is anticipated that onein eight people currently alive will eventually die of cancer. Cancermanifests itself in a wide variety of forms, characterized by differentdegrees of invasiveness and aggressiveness. Malignant tumors are thesecond leading cause of death in the United States, after heart disease.87% of cancer diagnoses in the U.S. are in people age 50 and older. Theglobal population is rapidly aging. Currently, 566 million people areaged 65 years old worldwide, with estimates of nearly 1.5 billion by2050, particularly in developing countries. Infections constitute athird of mortality in people over 65 years old. Moreover, lengtheninglife spans correlate with increased time in hospitals or long-term carefacilities and exposure to drug-resistant pathogens. The risk ofnosocomial infections increases with age, independent of duration spentin healthcare facilities. One theory is that as a person ages, theirimmune system changes and is less robust in addressing bacterialinfections. By enhancing the microbiome of a person as they age, it isbelieved that infections that would otherwise be encountered will beavoided, or at least the frequency and severity of the same will bedecreased. Aging is a pathophysiological phenomenon that is possible toinfluence so as to delay or postpone the aging process, and in turn,delay the onset and incidence of various cancers. The prospect ofachieving a longer life, free of age related functional decline andfragility, such as cancer, is perhaps the quintessential long-felt butunsolved problem of mankind. Increasingly, there is a growing awarenessof the importance of the variation in the gut microbiota as its affectsthe etiology of several age-related diseases, including cancer.

In the US, bladder cancer is the fourth most common type of cancer inmen and the ninth most common cancer in women. Non-muscle invasivebladder cancer (NMIBC) begins and stays in the cells lining the bladderwithout growing into the deeper main muscle layer of the bladder, andaccounts for the majority (70-80%) of patients diagnosed with bladdercancer. Bladder cancer has the highest recurrence rate of anymalignancy. Although NMIBC is a relatively benign disease, it recurs in50-70% of patients, of which 10-20% eventually progress to high-grademuscle-invasive disease. More than 1 million patients in the US andEurope are estimated to be affected by the disease.

Non-alcoholic fatty liver disease is a condition ranging from benignlipid accumulation in the liver (steatosis) to steatosis combined withinflammation. The latter is referred to as non-alcoholic steatohepatitis(NASH). NASH is viewed as the hepatic component of metabolic syndrome.Estimates from the USA are that 5.7% to 17% of all adults have NASH,while 17% to 33% of Americans have NAFLD. As obesity and insulinresistance reach epidemic proportions in industrialized countries, theprevalence of both NAFLD and NASH is increasing and is thereforeconsidered to be a major health hazard. Steatosis alone is considered arelatively benign condition for the liver itself and is also areversible condition. However, the transition towards NASH represents akey step in the pathogenesis, as it sets the stage for further damage tothe liver, such as fibrosis, cirrhosis and liver cancer. While themechanisms leading to steatosis are well described, little is knownabout the actual risk factors that drive hepatic inflammation during theprogression to NASH. Consequently, therapeutic options are poor.

Cachexia is a positive risk factor for death, meaning that if a patienthas cachexia, the chance of death from the underlying condition isincreased dramatically. Skeletal muscle atrophy is a nearly universalconsequence of cancer. Cachexia is considered the immediate cause ofdeath of a large proportion of cancer patients, ranging from 22% to 40%of cancer patients. The pathogenesis of cancer cachexia is poorlyunderstood. Only limited treatment options exist for patients withclinical cancer cachexia. Current treatment strategies involveattempting to improve an individual's appetite using appetite stimulantsand protein supplementation to provide the individual with requirednutrients. The reversal of cancer cachexia and muscle wasting leads toprolonged survival, and with the ability to retain muscle mass andstrength, it is believed that various forms of cancer treatment may bemore effective, if only due to the fact that the cancer victim may beable to withstand the rigors of the various cancer treatments involved.Cachexia was overlooked for many years, with doctors directing theirattention to the primary illness instead. Many, however, now viewcachexia as a distinct, treatable condition.

Probiotics are so-called “good” microorganisms (typically bacteria) thatare ingested (or contacted with a person) alive by an individual so thatthe introduced microorganisms can colonize the GI tract of the person.Conventional prebiotics are ingestible ingredients that selectivelysupport the growth or survival of the “good” microorganisms which aredesirably present in the GI tract. Conventional prebiotics are typicallya nutrient source (e.g., fructooligosaccharide orgalactooligosaccharide) that can be assimilated by one or more membersof the GI microbiome, but which are not digestible by the human host.

The development of molecular techniques to identify and quantifymicrobial organisms has revolutionized the microbial world. Genomiccharacterization of bacterial diversity relies on sequence analysis ofthe 16S ribosomal RNA gene, which is present in all bacteria andarchaea. The 16S rRNA gene contains species-specific hypervariableregions, which allow taxonomic classification, and highly conservedregions, which act as a molecular clock and a binding site for PCRprimers. Using current technologies, an organism does not need to becultured to determine its type by 16S rRNA sequencing.

The human gut is perhaps one of the most complex networks in the bodyand is colonized by trillions of microorganisms including bacteria,archaea, fungi, protists, and viruses, among which bacteria are themajor inhabitants. Hepatocellular carcinoma (HCC) is one of the mostcommon malignancies in the world. Gut microbiota has been demonstratedto play a critical role in liver inflammation, chronic fibrosis, livercirrhosis, and HCC development through the gut-liver axis. Gut microbialdysbiosis accompanies the progression of alcoholic liver disease,non-alcoholic fatty liver disease and liver cirrhosis, and promotes HCCprogression. Microbial dysbiosis contributes to cancer susceptibilityvia multiple pathways. Further studies have suggested that themicrobiota and their associated metabolites are not only closely relatedto carcinogenesis by inducing inflammation and immune dysregulation,which lead to genetic instability, but also interfere with thepharmacodynamics of anticancer agents. Chronic inflammation has beenverified as a driving cause of cancer. Inflammation promotes tumorprogression and accelerates the invasion and metastasis. The generationof inflammation-associated factors can also inactivate tumor-suppressorgenes (e.g., P53 mutation). The hepatic environment is greatlyinfluenced by the pathogens or metabolites produced by the microbiota inthe GI tract through the hepatic portal venous system. Liver exerts anessential effect on the host microbial community by filtering the bloodstream as well as metabolizing and neutralizing toxins derived fromintestinal microbes. Gut microbial dysbiosis contributes tohepatocarcinogenesis because the microbiota and microbial metabolitesare detected by liver resident immune cells and are able to modifyhepatic metabolism. NAFLD is considered to be a major risk factor forHCC.

The use of checkpoint inhibitors has revolutionized various cancertreatments. Improving the outcomes of such treatments by anunderstanding of how a person's microbiome can be beneficiallymanipulated to advance positive outcomes that employ checkpointinhibitors is a long sought but unsolved issue.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a method for treatingan individual suffering from one of bladder cancer and colorectal cancerthat involves the use of a clustered regularly interspaced shortpalindromic repeats (CRISPR) CRISPR associated protein (Cas) system or aCRISPR from Prevotella and Francisella 1 (Cpf1) to accomplish theselective killing or reduction in the number of a pathogenic bacteriawithin the individual, followed by the administration of an immunecheckpoint inhibitor.

The microbiota inhabiting our bodies influence cancer predisposition andetiology. The largest microbial community in the human body resides inthe gut and comprises somewhere between 300 and 1000 different microbialspecies. The human oral microbiome and the bacteria inhabiting suchmicrobiome are, in certain circumstances, also effective as agents inthe treatment of cancer. Various embodiments of the present inventioninvolve the modification of at least two, if not three separatemicrobiomes of a person to treat certain conditions. For example, thetreatment for cachexia may be achieved via modification of anindividual's oral microbiome via the delivery of particular bacteriadesigned to produce therapeutic amounts of tomatidine. The simultaneousprovision of bacteria to the individual's gut microbiome that aredesigned to produce therapeutic amounts of p53 protein can also beachieved, with the two separate microbiomes being employed to addressseparate but related issues involved in cancer treatments. Thisparticular aspect of the present invention, while simple in nature, isbelieved to have profound effects in avoiding undesired druginteractions that can complicate treatment regimens. By having differentmicrobiomes of the same individual administer different desiredcompounds, drugs, factors, proteins, etc. to the person's body, theability to separately control administration and amounts (as well as toaddress issues by killing bacteria in one but not the other microbiome)is rendered feasible as a way to administer desired cancer fightingagents to an individual.

In particular embodiments, the pathogenic bacteria is selected from thegroup consisting of E. coli, Pseudomonas aeruginosa and Klebsiellabacteria and the immune checkpoint inhibitor selected from the groupconsisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514,STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C,AUR-012 and STI-A1010. In other embodiments, one enhances the growth ofa second bacteria in the individual selected from the group consistingof Akkermansia, Bacteroides, Bifidobacterium, Clostridium, Enterococcus,Fusobacterium, LactoBacillus, Propionibacterium, Ruminococcus,Veillonella, Prevotella, Escherichia and Streptococcus bacteria.

Certain aspects of the present invention are directed to modifying aperson's intestinal (gut), oral or skin microbiota using specificcombinations of pre-biotics, pro-biotics and/or anti-biotics toestablish a defined microbiota that can treat and/or reduce thelikelihood that individuals will experience various diseases, includingcancer. The employment of various bacteria, whether in particularcombinations or after being modified using CRISPR-type systems, formsvarious embodiments of the present invention, and leads to improvedoutcomes when checkpoint inhibitors are used to treat various forms ofcancer.

For example, various embodiments of the present invention are directedto averting or reducing the likelihood of cancer by employing bacteriamodified to address p53 deficiency. In such a manner, rather thantreating human cells and the consequent issues surrounding geneticmanipulation of human cells for treatments of cancer, the presentinvention provides a method and system that employs the microbiome of aperson, whether than be oral, gut or skin, or a combination thereof, totreat cancer by increasing the level of p53 to take advantage of therole of such protein in the progression of various cancers. Provision ofmodified bacteria as described herein to pre-treat a person prior to acancer treatment, such as radiation, can also be used to lessen theotherwise detrimental effects of the radiation treatment. Moreover,after such treatments, provision of such modified bacteria to restorethe person's microbiomes, whether they be oral, skin or intestinal, isone aspect of the present invention. Use of modified skin bacteria totreat melanoma is one aspect of the present invention, thus providing away to treat skin cancer by providing essential compounds to reduce thespread and health of cancer cells while at the same time, enhancing thegrowth and propagation of beneficial bacteria, especially those modifiedas described herein via a CRISPR system.

Through coevolution of bacteria, archaea and fungi with the human hostover thousands of years, a complex host-microbiome relationship emergedin which many functions, including metabolism and immune responses,became codependent. This coupling becomes evident when disruption in themicrobiome composition, termed dysbiosis, is mirrored by the developmentof pathologies in the host. Among the most serious consequences ofdysbiosis, is the development of cancer. Various embodiments of thepresent invention are directed to the field of Oncology, and inparticular, embodiments directed to a method of ameliorating, treating,or preventing a malignancy in a human subject wherein the steps of themethod assist or boost the immune system in eradicating cancerous cells.In certain embodiments, administration of beneficial bacteria to anindividual's microbiome that have been modified so as to produceeffective amounts of desired compositions, compounds, agents, e.g.tomatidine, p53 protein, etc., is employed to address cancerousconditions. In several embodiments, the administration of suchbeneficial bacteria and microbes to an individual's microbiome invokeseither an active (or a passive) immune response to destroy, weaken orrender less invasive certain cancerous cells, and preferably maintainsmuscle tissue to combat cancer cachexia. Various embodiments of thepresent invention involve the expression/production by microbes of anindividual's microbiome of a phytochemical to enhance the lifespan andhealth of a human.

Preferably, the modified bacteria employed in the present invention areadministered orally to a patient in order to deliver the therapeuticdirectly to the site of inflammation in the gut. Suppositories can alsobe employed for administration of particular bacteria that may be moredifficult to deliver to a particular portion of a person's body, e.g.those that may be destroyed while passing through a person's stomach.The advantage of an oral or rectal approach is that it avoids systemicadministration of immunosuppressive drugs and delivers the therapeuticdirectly to the gastrointestinal tract. In certain embodiments, theviability and stability of such modified bacteria is enhanced to supportthe production of such microbes of desired agents/compounds, e.g.tomatidine, p53 protein, rapamycin, resveratrol, methylene blue, etc.and by doing so, a method is provided that reduces gut inflammation,enhances gut barrier function, and/or treats autoimmune disorders.Preferably, such modified bacteria are capable of producing therapeuticanti-inflammation and/or gut barrier enhancer molecules, particularly inthe presence of reactive nitrogen species, and more preferably thebacteria are functionally silent until they reach an environmentcontaining local RNS, wherein expression of the therapeutic molecule isinduced. In certain embodiments, the genetically engineered bacteria arenon-pathogenic and may be introduced into the gut in order to reduce gutinflammation and/or enhance gut barrier function. For example, in someembodiments, the bacteria are under the control of a RNS-responsiveregulatory region and a corresponding RNS-sensing transcription factorsuch that a desired product, e.g. butyrate is produced, which inducesthe differentiation of regulatory T cells in the gut and/or promotes thebarrier function of colonic epithelial cells. Short-chain fatty acidproduction by commensal bacteria is important in regulating the immunesystem in the gut. Butyrate plays a direct role in inducing thedifferentiation of regulatory T cells and suppressing immune responsesassociated with inflammation. Butyrate is normally produced by microbialfermentation of dietary fiber and plays a central role in maintainingcolonic epithelial cell homeostasis and barrier function. Use of suchmodified bacteria, especially those modified via CRISPR-Cas systems,provides a way to generate a desired therapeutic effect in a manner thatlowers the safety issues associated with systemic exposure. Resveratrol(3,4′,5-trihydroxystilbene; C.sub.14H.sub.12O.sub.3) is a polyphenolicphytoalexin found in grapes, berries, peanuts, and wines. Resveratrolhas been viewed as an antioxidant, anti-inflammatory, anti-apoptotic,and anticancer agent. Moreover, it has been reported that resveratrolmodulates mitochondrial function, redox biology, and dynamics in both invitro and in vivo experimental models. Resveratrol also attenuatesmitochondrial impairment induced by certain stressors. Resveratrolupregulates, for example, mitochondria-located antioxidant enzymes,decreasing the production of reactive species by these organelles.Resveratrol also triggers mitochondrial biogenesis, ameliorating themitochondria-related bioenergetics status in mammalian cells. Braincells (both neuronal and glial) are susceptible to mitochondrialdysfunction due to their high demand for adenosine triphosphate (ATP).Additionally, brain cells consume oxygen (O.sub.2) at very high rates,leading to a proportionally high mitochondrial production of reactivespecies. One aspect of various embodiments of the present invention isthe maintenance of mitochondrial function in various cell types toaddress degenerative diseases, which involve mitochondrial impairmentand increased generation of reactive species, leading, for example, toneuroinflammation and cell death. The mechanism by which resveratrolprotects mitochondrial function and dynamics is not completelyunderstood, but it is known that resveratrol is able to inducecytotoxicity depending on its dosage. Resveratrol produced by themicrobiome of an individual can be employed to improve the dysregulationof the gut microbiota induced by a high-fat diet, as it will result inincreasing the ratio of Bacteroides-to-Firmicutes and also increases thegrowth of LactoBacillus acidophilus and Bifidobacterium in humans. It isbelieved that resveratrol modifies the intracellular environment bychanging the oxidizing milieu into a reducing milieu and upregulatesintracellular glutathione, potentiating a signal transduction cascadethat results in mitophagy, and thus paves the way to an anti-agingenvironment. Rapamycin was first discovered in Easter Island soilbacteria in the 1980s. It is known that rapamycin extends the life spanof mice. The protein that rapamycin targets is a kinase called mTOR.This kinase plays a role in a variety of pathways. mTOR suppresses somesenescent cells from secreting their cocktail of problematic moleculesand mTOR plays a role in the positive effects of caloric restriction.But given the disparity of microbiome constituents between any twoindividuals, the present inventors contend that the manner by which toeffectively address aging of any particular individual lies in takingadvantage of the noted differences of each individual's microbiome toaddress the aging mechanisms involved.

One aspect to the present invention relates to the fact that microbiomecomposition can influence chemotherapy efficacy. Various bacterialstrains have been found to enhance resistance against pathogenicinfections and to improve the therapeutic efficacy of immune checkpointinhibitors. The effects of the intestinal microbial metabolite butyrateinterferes with the development of colorectal cancer. Increased levelsof SOFA, namely, acetate, butyrate, and propionate in CRC are usuallylinked with lower risk and improved prevention or therapy.Microbial-derived butyrate counteracts tumor development. Butyratesuppresses proinflammatory genes and tumor growth, the latter viahistone deacetylase inhibition, which downregulates oncogenic signalingpathways. However, depending on local concentration, SCFAs can also playa dual role in cancer. Butyrate, for instance, also inhibitsproliferation of healthy intestinal progenitor cells. Certain strainsare associated with cancer. Porphyromonas gingivalis has been found tobe involved in several different types of cancer. A significantoverabundance of P. gingivalis was found in fecal samples from CRCpatients. Similarly, Fusobacterium, Peptostreptococcus, Prevotella,Parvimonas, Bacteroides, and Gemella are among the most prominentCRC-associated bacteria. Moreover, certain metabolites are known toenhance cancer. Exogenous formate may fuel cancer invasion as increasedformate overflow is a hallmark of oxidative cancer. One aspect of thepresent invention is therefore directed to shifting the metabolic stateof the tumor microenvironment into a lesser oncometabolite-containingstate.

Certain embodiments involve the administration of beneficial bacteria toan individual's microbiome that have been modified so as to produceeffective amounts of desired compositions, compounds, agents, etc., e.g.tomatidine, p53 protein, rapamycin, resveratrol, methylene blue,butyrate, SCFA's, etc. For example, in several embodiments, theadministration of beneficial bacteria and microbes to an individual'smicrobiome invokes either an active (or a passive) immune response todestroy, weaken or render less invasive certain cancerous cells. Variousother embodiments are drawn to the co-administration of one or more oftomatidine, p53 protein, rapamycin, resveratrol, methylene blue etc., incombination with conventional therapies for treating diseases, such ascancer. In particular, the co-administration of various pre-bioticcompositions to enhance and sustain the desired effects of thebeneficial modified bacteria forms another aspect of the presentinvention. In this regard, incorporation by reference of U.S. Patentpublication No. 20160213702 to Maltzahn et al. is included as part ofthe written description of various aspects of the present invention. Forexample, in view of the fact that the microbiota of humans is complexand varies by individual depending on genetics, age, sex, stress,nutrition and diet, modifying the numbers and species of gut, oral,vaginal and skin microbiota can alter community function and interactionwith the host. A number of probiotic bacteria known in the art, as wellas some foods considered to be ‘prebiotic’ that contain substances thatpromote the growth of certain bacteria and that stimulate beneficialmicrobiota shifts to improve human health, can be employed in concertwith the modified bacteria as described herein to effect desired cancertreatment regimens. For example, the administration of glycans in anamount effective to modulate the abundance of the bacterial taxa can beused to achieve better outcomes in the treatment of various age relateddiseases, including cancer.

One aspect of the present invention relates to the use of variousLactoBacillus species to reduce LDL, cholesterol, and triglycerides tocause an improvement and amelioration of inflammation andsteatosis—which can lead to cancer.

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for colorectalcancer. NAFLD is associated with a high incidence of CRC. Age is animportant factor for CRC and the CRC incidence increases with age. Thepresent inventors believe that particular modulation of the gutmicrobiome, including the establishment and maintenance of certainbeneficial bacteria, including LactoBacillus, Bifidobacterium, andcertain Streptococcus species, forms the basis of a treatment of NAFLD,as well as NASH, and in particular, the use of particular species thathave been modified via a CRISPR system. Nonalcoholic steatohepatitis(NASH) is a more advanced form of NAFLD where liver injury has occurred,and can lead to liver failure, portal hypertension, hepatocarcinoma andcirrhosis. Even without significant changes in BMI, glucose, or LDL2,probiotic use is believed to significantly decrease ALT, AST, totalcholesterol, HDL, and TNF-.alpha.1.

Thus, in various embodiments of the present invention, the employment ofparticular probiotics as described herein, provides a treatment forNAFLD that shows improvements in intestinal dysbiosis, leading todecreasing intestinal permeability, endotoxemia and subsequentinflammation.

The most frequent cause which leads to obesity is a dysbalance betweenenergy intake and energy expenditure. The gut microbiota contributes tohost metabolism. Gut microbiota not only influence absorption anddisposal of nutrients to the liver, but also can lead to the developmentof “metabolic endotoxemia” and activation of TLR ligands, which canstimulate liver cells to produce proinflammatory cytokines, therebyinitiating inflammation and fibrogenesis, which characterize NASH.Another possible molecular mechanism implicated in NAFLD development isthe alteration in LPS-endocannabinoid (eCB) system regulatory loops andbile acid metabolism. Thus, certain embodiments of the present inventionare directed to the modification of intestinal bacterial flora byspecific probiotics to achieve a therapeutic approach for the treatmentof NAFLD.

One strategy for NAFLD treatment encompassed by the present inventionrelates to a treatment for obesity that involves manipulation of anindividual's gut microbiota. Thus, modulation of gut microbiota byprobiotic treatment or dietary intervention provides beneficial effectswith respect to body weight, influence on glucose and fat metabolism,insulin sensitivity and reduction in chronic systemic inflammation, allof which can impact the status of NAFLD. Probiotic positive effects onhost metabolism are specifically directed to beneficial levels ofLactoBacillus and/or Bifidobacterium strains. For example, employment ofSaccharomyces cerevisiae var. boulardii, Enterobacter halii orAkkermansia muciniphila are used to achieve beneficial effects forobesity and NAFLD. In certain embodiments, because obstructive sleepapnea and attendant fatigue are common in patients with NAFLD, oneaspect of the present invention relates to the use of “no-snore strips”as described herein (and in more extensive pending patent applicationsincorporated herein by this reference, e.g. U.S. Pat. No. 9,445,936)such that use of such strips can beneficially modify not only thepopulations of oral bacteria, but also snoring patterns, thus providingthose suffering from NAFLD with a way to manage such condition to permitthem to address fatigue issues and to thus sleep better, exercise more,etc.

Gut bacteria alter the way individuals store fat, how levels of glucoseare balanced in the blood, and how humans respond to hormones that makeindividuals feel hungry or full. Certain population mixes of microbesset the stage for NAFLD, obesity and diabetes. The gut community in leanpeople is diverse while obese people have a gut microbe community thatis comparatively less diverse. Lean individuals, for example, tended tohave a wider variety of Bacteroidetes, a population of varied microbesthat specialize in breaking down bulky plant starches and fibers intoshorter molecules that the body can use as a source of energy.

Probiotics have physiologic functions that contribute to the health ofgut microbiota, can affect food intake and appetite, body weight andcomposition and metabolic functions through gastrointestinal pathwaysand the modulation of the gut bacterial community. Thus, in variousembodiments of the present invention, probiotics are employed, e.g.(Enterococcus faecium, Streptococcus thermophilus L. acidophilus,Bifidobacterium longum, L. plantarum and/or B. lactis) to significantlyreduce total serum cholesterol and LDL cholesterol and to improve theLDL:HDL cholesterol ratio. In particular embodiments, a CRISPR-Cassystem (Clustered Regularly Interspaced Short PalindromicRepeats-CRISPR-associated) is employed to alter one or more of thesebacteria to modify various virulence factors associated with bacteria sothat beneficial populations of bacteria inhabit an individual's oraland/or gut microbiome.

Various embodiments of the present invention relate to a compositioncapable of increasing the level of anti-oxidized low-density-lipoprotein(oxLDL) antibodies in vivo for use in the treatment or prevention ofNASH. OxLDL is an immunogenic molecule that stimulates the induction ofanti-oxLDL antibodies. Phosphorylcholine, a component of Streptococcuspneumoniae, is a major antigen in oxLDL, which is recognized byanti-oxLDL antibodies that have protective properties. One embodimentrelates to the expression of OxLDL in bacteria via employment of aCRISPR-Cas system to insert genes for OxLDL such that such modifiedbacteria produce OxLDL to therefore stimulate the induction ofanti-oxLDL antibodies, thus providing the protective effects of suchantibodies. Using the present invention, fibrosis can be decreased orprevented by the production and administration of anti-oxLDL antibodiesto avoid inflammation of the liver and to therefore treat NASH andNAFLD. While antibodies against oxLDL are known in the art, variousembodiments of the present invention relate to a new medical use of suchantibodies, as well as to methods and systems that modify gut bacteriato enhance the production of such antibodies. In other words, variousembodiments of the invention relate to a composition comprisingantibodies against oxLDL for use in the treatment or prevention ofhepatic inflammation or more in particular the treatment or preventionof NASH, and/or the use of oxLDL antibodies for the preparation of amedicament for the treatment or prevention of hepatic inflammation andin the treatment of NASH. In certain embodiments, a method of treatmentor prevention of hepatic inflammation is provided where oxLDL antibodylevels are increased by modification of particular bacteria using aClustered Regularly Interspaced Short PalindromicRepeats-CRISPR-associated system (CRISPR-Cas) or Clustered RegularlyInterspaced Short Palindromic Repeats from Prevotella and Francisella 1(CRISPR/Cpf1) system so that the bacteria is able to produce desiredlevels of oxLDL antibodies.

In other embodiments, the methods and systems disclosed herein aredirected to modifying the gut microbiota of an individual to amelioratethe progression of NAFLD, including reducing liver aminotransferases,total-cholesterol, TNF-.alpha. and improving insulin resistance inindividuals with NAFLD. In certain embodiments, NAFLD is thus treated bymodulation of the gut microbiota. Effective treatments include employinga method of populating a subject's gastrointestinal tract with a diverseand useful selection of microbiota in order to alter a dysbiosis.Various aspects and embodiments of the invention are directed to methodsand compositions for modulation of NAFLD of an individual's gutmicrobiome by using bacteria that have been treated with a CRISPR-Cas orCRISPR-Cpf1 system to reverse antibiotic resistance or to renderineffective certain virulence factors in pathogenic bacterial cell, aswell as modifying gut bacteria in a manner to make them “better” invarious ways, including an ability to outcompete other undesiredbacteria. Other various embodiments of the present invention relate tothe employment of engineered autonomously distributed circuitscontaining programmable nucleases (e.g. “programmable nucleasecircuits”) that are delivered to microbial organisms in vivo to modulatethe expression of certain antibiotic resistant and virulence factors ofparticular microbial organisms. Some embodiments employ the Type IICRISPR-Cas (Clustered Regularly Interspaced Short PalindromicRepeats-CRISPR-associated) system of Streptococcus pyogenes to reverseantibiotic resistance in a wide range of microbial organisms. In certainembodiments, the CRISPR-Cas system is used to weaken resistance ofmicrobial pathogens to existing antibiotics. The use of the CRISPR-Cassystem may be viewed as a paradigm shift in combating pathogens becauseit enables autonomous and distributed neutralization of disease at thegene level. Various aspects of the present disclosure provide methodsthat comprise modifying bacterial cells to target a gene or nucleotidesequence of interest, and in particular, genes involved in the storageof fat. Such modified bacterial cells include an engineered autonomouslydistributed circuit having at least one nucleic acid encoding aprogrammable nuclease that targets a gene or nucleotide sequencedirected to fat metabolism.

While there are medications approved for treating diseases andconditions associated with NAFLD, there are currently no medicationsspecifically approved for the treatment of NAFLD itself. Treatmentprotocols have instead been focused upon the associated conditions, suchas the metabolic syndrome. Conventional treatment of NAFLD includesweight loss, restricting dietary fat, administration of medicationsemployed in the treatment of an associated condition and administrationof medications employed in the treatment of hyperlipidemia. Manymedications employed to treat conditions associated with NAFLD arehepatotoxic.

Various embodiments of the present invention are directed to a methodfor treating NAFLD in a subject in need thereof that includesadministering a composition including a therapeutically effective amountof Prevotella, and more preferably Prevotella that has been modified,e.g. by CRISPR-Cas, in a manner that reduces the effect of at least oneof the virulence factors of such bacteria. Other embodiments involve theemployment of bacteria of the Bacteroides family that have been modifiedto reduce the amount of a ligand-activated transcription factor.

Dysbiosis in a person's gut has a significant role in the pathogenesisof human NAFLD/NASH. In various embodiments of the present invention,administration of probiotics, as well as associated fiber diets tosupport such bacteria, is involved, in some embodiments employingBifidobacterium and LactoBacillus strains. Control of the bacterialflora lowers proinflammatory cytokine production (tumor necrosisfactor-.alpha., interleukin-6, interferon-.gamma.) via down-regulationof the nuclear factor kappa B, and decreases oxidative stress.Probiotics can reduce the urease activity of bacterial microflora,decrease fecal pH value and reduces amino-acid fermentation and ammoniaadsorption; reduce aminotransferases, and improve the lipid status inNAFLD patients. Each of these may be modified via CRISPR-Cas systemsemployed to alternative characteristics of an individual's microbiome.

Microbiome research in liver disease has evolved recently as an excitingnew field. Prebiotics encompass products that promote the growth ofbeneficial intestinal microbiota. Probiotics include live microbialstrains in predefined quantities. Both prebiotics and the use ofprobiotics is involved in the various embodiments of the invention asherein described. The present invention is directed in variousembodiments directed to ways to modify the microbiota to treat hepaticsteatosis, liver inflammation, fibrosis, and developing and advancedliver disease. The purposeful manipulation of the gut microbiota is doneto address various liver diseases at both early and late disease stages.

More than 90% of the adult microbiome is composed of species belongingto four bacterial phyla: Firmicutes, Bacteroidetes, Actinobacteria, andProteobacteria. Differences exist, however, with respect to differentindividuals as well as in different habitats. For example, Firmicutesare the major species in the intestine, vagina, skin, and oral cavity,while Actinobacteria and Proteobacteria are more dominant in the oralcavity, skin, and nasal cavity. The enterotype is a classification ofthe microbiome, with the gut microbiome being classified into threeenterotypes. Each enterotype includes a dominant species selected fromthe group consisting of: Bacteroides, Prevotella, and Ruminococcus, withenterotypes being unrelated to race, residential region, or diet.

It is believed that commensal microbiota protect against biliary injuryand liver fibrosis. The present inventor believes that there is asignificant association of fatty liver with H. pylori infection. Thus,various embodiments involve the modification of an individual'smicrobiome, including H. pylori in one's stomach, to combat NAFLD andNASH and cancers related thereto. Thus, use of CRISPR-Cas to render H.pylori more susceptible to particular antibiotics is one way in whichsuch modification may be achieved.

NAFLD is a complex disease and a treatment targeting one pathologicalprocess often also causes changes in other pathways. Prebioticsrepresent a specific type of dietary fiber that when fermented, mediatemeasurable changes within the gut microbiota composition, usuallycausing an increase in the relative abundance of bacteria thought of asbeneficial, such as bifidobacteria or certain butyrate producers.Prebiotics are usually non-digestible carbohydrates, oligosaccharides orshort polysaccharides, including inulin, oligofructose, galactofructose,galacto-oligosaccharides and xylo-oligosaccharides, all leading toincreasing the relative abundance of bifidobacteria and lactobacilli.The gut of individuals with various maladies, including obesity, harborbacteria in their gut that establishes an inflammation-associatedmicrobiome, often providing a lower potential for butyrate productionand reduced bacterial diversity. Thus, one objective of the presentinvention is to alter the microbiome of such individuals to increasebacterial diversity in their gut and to increase levels of butyrateproduction. Patients with NAFLD have small intestinal bacterialovergrowth and increased intestinal permeability. Thus, altering themicrobiome of such individuals is achieved to counter the progression ofNAFLD. In certain embodiments, one objective is to increase theproportion of Ruminococcaceae in a person's microbiome and to alsoreduce the proportion of Escherichia, e.g. by modifying Escherichia viaCRISPR-Cas to make it less viable than it otherwise would be.

Probiotics can reduce liver aminotransferases, total cholesterol, tumornecrosis factor .alpha. and improve insulin resistance in patients withNAFLD. Similarly, treatment of other diseases in the gut, likeinflammatory bowel disease (IBD) is implicated with respect tomodification of the gut microbiome. The concept of an altered gutmicrobiota or dysbiosis is possibly the most significant development inIBD and NAFLD research in the past decade. A definitive change of thenormal gut microbiota with a breakdown of host-microbial mutualism isbelieved to be the defining event in IBD and NAFLD development.

In other embodiments, one objective is to increase the levels ofLactoBacillus, Leuconostoc, Lactococcus, Pediococcus and Firmicutes inan individual's gut microbiome, while reducing the levels ofBacteroidetes and Akkermansia spp. In certain other embodiments, oneobjective is to increase the levels of Prevotella and Roseburia (abutyrate-producer) in a person's gut microbiome, and especially thecolon microbiome. Other embodiments focus on increasing the levels ofBacteroides in the person's gut and decreasing the levels ofEscherichia, Lachnospiraceae and Megasphaera.

Periodontal disease is a chronic infectious disease of the tissuessurrounding the teeth that result in tooth loss. Several reports haveindicated that periodontal infection is related to NAFLD. Both NAFLD andperiodontal disease are chronic inflammatory conditions that are knownas ‘silent diseases’. Therefore, both conditions need to be detectedearly and treated under collaborative medical and dental care in orderto prevent progression to NASH. The prevalence of NAFLD in the Americangeneral adult population is 10%-40% and that of NASH is approximately2%-5%. One aspect of the present invention is directed to therelationship between periodontal pathogens, e.g. composed of P.gingivalis, and the severity of NAFLD. The eradication of periodontalpathogens, such as P. gingivalis infection, is believed to have abeneficial effect upon NASH.

Certain embodiments of the present invention are directed to a methodfor treating non-alcoholic fatty liver disease by providing to anindividual in need thereof an effective amount of a compositioncomprising modified L. reuteri bacteria, preferably using CRISPR-Casand/or Cpf1 systems, to provide such bacteria in a manner so that theyhave the ability to survive the conditions in the duodenum or jejunum ofthe small intestine. Other embodiments involve a method for treatingnon-alcoholic fatty liver disease involving establishing in the gut ofan individual a population of beneficial bacteria selected from thegroup consisting of LactoBacillus, Bifidobacterium, and Streptococcusspecies and administering at least 6 grams per day of fiber to theindividual to maintain the beneficial bacteria in the gut of theindividual. Still other embodiments are directed to a method fortreating non-alcoholic fatty liver disease by increasing oxLDL antibodylevels in an individual by modifying bacteria, preferably using aCRISPR-Cas or Cpf1 system, so that the bacteria is able to producedesired levels of oxLDL. Yet other methods involve the modulation ofNAFLD of an individual's gut microbiome by using beneficial bacteria,e.g. such as one or more of bacteria from one or more of the phylas:Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria,preferably treated with a CRISPR-Cas or CRISPR-Cpf1 system to reverseantibiotic resistance or to render ineffective certain virulence factorsin pathogenic bacterial cells. In other embodiments, an individual isadministered a therapeutically effective amount of Prevotella, and morepreferably Prevotella that has been modified in a manner that reducesthe effect of at least one of the virulence factors of such bacteria.Certain embodiments are directed to a method for treating non-alcoholicfatty liver disease involving the modifying of bacteria of theBacteroides family so that they produce reduced amounts of aligand-activated transcription factor as compared to non-modifiedbacteria. In preferred embodiments, probiotics are further provided tofeed such bacteria, with the result being improvements in levels ofdensity lipoprotein, and tumor necrosis factor-.alpha.

Other aspects of the present invention are directed to treating and/orreducing the likelihood of colorectal cancer. Humans possess aninflammatory response—the triggering of the overproduction ofhydrochloric acid—as the stomach's primary response to bacterialcolonization. Inflammation of the stomach lining coincides withproduction of peptides called cytokines, which stimulate production of ahormone called gastrin. Gastrin triggers parietal cells in the stomachlining to produce more hydrochloric acid, which kills off most invadingmicrobes. Notably, H. pylori is the only bacterial organism in thestomach that cannot be killed by hydrochloric acid. If you inhibitgastric acid production, you interfere with the stomach's naturaldefense mechanism. An abnormally low level of acidity in the stomach isa factor in various disease states. Since reduced gastric acidity doesappear to make the mammalian stomach more vulnerable to bacterialinvasion and gastritis, however, physicians are advised to re-evaluatethe long-term use of proton-pump-inhibiting drugs in their patients.

One aspect of the several embodiments of the present invention isdirected to the modification of microbes in a manner that reduces, ifnot eliminates, the symptoms of GERD. Helicobacter pylori is one ofseveral pathogens that persist within the host despite a robust immuneresponse. H. pylori elicits a proinflammatory response from hostepithelia, resulting in the recruitment of immune cells which manifestsas gastritis. Certain embodiments employ CRISPR-Cas or Cpf1 systems torender H. pylori more susceptible to certain drugs, includingantibiotics, thus addressing the resistance otherwise experienced bytreating H. pylori with antibiotics.

While not bound by theory, it is believed that H. pylori survivesantimicrobials, including calprotectin (CP), which employs nutrientsequestration, through alteration of its outer membrane. Thus, oneembodiment of the present invention relates to the interference with andmodification of the normal mechanism of H. pylori resistance toantibiotics by affecting the ability of H. pylori to form biofilms,including the retention and maintenance of lipid A production, which isnormally interfered with by H. pylori when contacted by CP. Inparticular embodiments, CRISPR-Cas systems are employed to undermine theability of H. pylori to form biofilms. One such strategy is to addadditional genetic components in H. pylori cultures that include lipid Aexpression and the purposeful inclusion of such a culture of H. pyloriin a person's stomach so as to establish a competitively beneficialculture of such modified H. pylori in the stomach. Thus, once suchbacteria are the predominant bacteria, as compared to non-modified H.pylori, then the application of CP (or other suitable antibiotics) canbe used to eliminate or vastly reduce the number of H. pylori bacteriain the person's stomach. Having the individual provided with further H.pylori cultures that possess such modified characteristics is furthercontemplated as a way in which to preclude the reestablishment of a wildtype H. pylori culture from persisting in the person's stomach. By sucha strategy, the person is able to substantially eradicate H. pylorispecies that are resistant to antibiotics. Other ways to decrease theformation of H. pylori biofilms include increasing cell surfacehydrophobicity. Another way is to enhance the function of the Lpx lipidA biosynthetic enzymes (e.g. LpxF, LpxL, and LpxR enzymes) to ensurethat their functions are not perturbed. Thus, to combat H. pyloriresistance to cationic antimicrobial peptides, one target is to affectthe formation of biofilms and to reduce the ability of H. pylori tomodify endotoxins. This can be achieved in various ways, but preferablyby employment of CRISPR-Cas systems to interfere with genes involved inthe formation of biofilms by H. pylori.

Certain embodiments are directed to the modification of resident H.pylori populations in vivo in a person's stomach so as to beneficiallydisrupt the colonization of the gastric glands by H. pylori. Otheraspects involve the modification of dietary components and essentialmicronutrients in concert with the gastrointestinal microbiota to affecta beneficial modification of H. pylori activity so as to maintain it asa commensal bacteria and to prevent its activity as a pathogen, thusprecluding its carcinogenic potential.

One aspect of the present invention is directed to therapeuticinterventions in the microbiome directed against molecular entities,such as essential and antibiotic resistance genes to quorum sensingsystems components used to control microbial networking behaviors,including the chemical communication and production of virulencefactors. Various embodiments are focused on dietary interventions andmicrobial modification genetic tools to modify and/or eliminatepathogenic microorganisms and to control dysbiosis. Various embodimentsof the present invention are also directed towards the modification ofthe human-microbiota ecosystem to promote health and to combat disease,including the modification and/or elimination of certain bacteria livingin the human body. The determination of human microbiota and theanalyses of the presence or absence of specific microbial species inaccordance with particular diseases provides one of skill in the artwith the ability to identify particular biomarkers and to target thesame to treat GERD.

While phage therapy could potentially have beneficial impact on humanmicrobiomes, host specificity greatly limits the types of bacteria thatcan be employed and the selection of a specific phage to use as atherapeutic agent requires in-depth knowledge of the pathogen causing agiven disease. In the absence of such knowledge, some have suggested theuse of a cocktail of different species of phages to broaden the range ofaction, but such a cocktail could have undesired negative effects on themicrobial community. Thus, in preferred embodiments of the presentinvention, CRISPR systems are employed to effect desired microbialmodifications. The relative simplicity of the mechanism of action andthe peculiarities of Cas9 make the CRISPR/Cas9 system an ideal tool fora vast assortment of procedures, particularly for genomic editing, andin various embodiments of the present invention, the editing ofbacterial strains is employed to interfere with the development of GERDand to otherwise treat GERD.

In various embodiments of the present invention, various targets forintervention using CRISPR-Cas systems include the modification ofbacteria resident in the human gut that are distinct from humans invarious respects. For example, most bacteria synthesize thiamine denovo, whereas humans depend on dietary uptake. Methionine is notsynthesized de novo in humans and must be supplied by diet. In contrast,most bacteria need to synthesize methionine to survive. There are amyriad of other orthologous gene groups conserved in both human andhuman commensal gut microflora that are not suitable targets for drugdevelopment. The majority of unique targets found in microbes' genomesare genes responsible for the metabolism of carbohydrates, amino acids,xenobiotics, methanogenesis, and the biosynthesis of vitamins andisoprenoids, and in particular for the purposes of various embodimentsof the present invention, focus is directed to those genes that arenon-homologous to those encompassed in human genome. A number ofmicrobial genes and products, including bacteriocins, lysins, holins,restriction/modification endonuclease systems, and other virulencefactors contribute to resistance to antibiotics. Thus, an alternative tokilling or inhibiting growth of pathogenic bacteria is targeting thesekey regulatory systems. Other aspects of the present invention aredirected to targeted changes in microbiota by the rational use ofprebiotics and probiotics to abolish metabolic alterations associatedwith various maladies, including GERD, obesity, cancer, etc.

In particular embodiments, compounds such as halogenated furanonesproduced by many microbial species, mostly belonging to theproteobacteria, are employed to interfere with AHL and AI-2 QS pathwaysin Gram-negative and Gram-positive bacteria. It is believed that byinterrupting normal systems of bacterial inter and intra quorum sensing,one may effectively modify bacterial cell-cell communication in a mannerthat prevents colonization by pathogenic bacteria, and in particular,can be employed to interfere with biofilm formation by H. pylori andthus, treat GERD.

High doses and frequent use of antibiotics can disrupt and destabilizethe normal bowel microbiota, predisposing patients to developClostridium difficile infections. Up to 35% of these patients develop achronic recurrent pattern of disease. Fecal bacteriotherapy is thetransplantation of liquid suspension of stool from a donor (usually afamily member) and has been used successfully in severe cases ofrecurrent C. difficile relapse. Many problems exist with this therapysince it can increase the risks of transmitting other pathogens. Oneparticular focus of the present invention is to employ transplantedmicrobiota to treat metabolic disorders in humans but that limit therisks involved in conventional fecal transplants. For example, via theuse of modified bacteria (e.g. using CRISPR-Cas systems) one is able tomore effectively employ antibiotics to target particular regions of thebody, to target particular bacteria, etc. in a manner that avoids thecomplications experienced in the typical use of antibiotics, which causecomplications from C. difficile growth.

In particular embodiments, probiotic microorganisms that possessresistance to low gastric pH and have the capacity to reach theintestines alive, are used to exert beneficial effects on the humanbody, preferably lactic acid-producing bacteria of the LactoBacillus andBifidobacterium genera. Such microorganisms are preferably thosemodified using CRISPR-Cas to provide a population that can be moreeasily controlled and manipulated to maintain particular levels in anindividual's microbiome. Specifically, some embodiments of the presentinvention include regulating the balance of intestinal microbiota byphysically blocking the adhesion of pathogenic species onto epithelialcells, such blocking action directly mediated by means of increases inthe production of a mucosal barrier by goblet epithelial cells and/or byregulating epithelial permeability by enhancing the formation oftight-junctions between cells. Use of CRISPR-Cas to modify or deletevirulence factors of particular bacteria, such as adhesion abilitiesthereof, is employed in this fashion.

In various embodiments of the present invention, CRISPR/Cas9 is used toselectively deplete a given bacterial community of a particular harmfulstrain or species, or of particular virulence factors possessed byparticular strains of bacteria. Thus, in certain embodiments, theidentification of a harmful pathogen is performed, and CRISPR/Cas9 isthen used to selectively deplete or modify that particular bacterialspecies from an individual's gut microbiota. The use of antibiotics isbelieved to increase the ability of bacteria to acquire drugresistance-encoding plasmids. Thus, the CRISPR/Cas9 system may be usedto introduce specific mutations into essential, antibiotic resistanceand virulence genes, as well as to directly modulate the expression ofparticular genes. For example, one can employ a Cas protein that lacksnuclease activity but retains a binding capacity so as to repressbacterial transcription by binding to promoter regions to effect theblocking of transcriptional initiation and/or elongation. CRISPR-Cas orCpf1 systems may also be used to fuse regulatory domains in order toswitch on/off the expression of specific genes. Thus, the presentinvention includes the engineering of commensal bacteria with improvedproperties using a CRISPR/Cas system to prevent and treat diseases. Oneof skill in the art will appreciate the steps required to affect thedesired levels of target specificity and delivering efficiency.

Still other embodiments employ the modification of various beneficialbacteria so that they express certain compounds and substances, notablythose substances found to be effective as an anti-H. pylori agent, suchas those isolated from garlic and ginseng. Alliin, the main activemolecule present in Garlic extract, is used to effect immune modulation.The use of dialkyl-thiosulfinate and/or propy thiosulfonate can beemployed to improve disease resistance of a pathogen, with thesecompounds generated from the natural degradation of propiin, a moleculepresent in most Allium species, and more specifically onion, shallots orchives. Still other sulfur compounds may be employed to inhibit H.pylori colonization. Using CRISPR-Cas, one is able to modify residentbacteria to express the active ingredient in garlic found to be aneffective killer of H pylori. Such expressed compounds include thosedescribed above.

Similarly, another aspect of the present invention is to control H.pylori populations in an individual's stomach by a diet includingkimchi. Other aspects are directed to the expression of kimchi genes byone or more bacterial species that reside in a human stomach. In amanner similar to the expression of certain genes derived from garlic,one is therefore able to control the population of H. pylori in aperson's stomach. The use of CRISPR-Cas to insert genes into particularbacteria so as to facilitate the control of H. pylori is one aspect ofcertain embodiments, including the insertion of genes having the activeagent contained in Korean kimchi and garlic. The incidence of gastriccancer is about 20 per 100,000 population (in Korea) and about 50 per100,000 population (in Japan, where far less kimchi is eaten),demonstrating that kimchi is effective as a cancer preventative agent.Similarly, the expression of garlic related genes by one or morebacterial species that reside in a human stomach is another embodimentof the present invention. The use of garlic (as well as kimchi) toaddress GERD is considered to be a teaching away from the prior art, asmany have identified garlic and onions as causing heartburn. Garlic(allium sativum), like onions, shallots and leeks, among others, belongsto the alliaceae family, and all contain organosulfur products. Garlicin particular contains allicin, an organosulfur compound that isproduced when garlic is broken or crushed, through the action of theallinase enzyme on alliin. Allicin is a potent phytocide, with markedantibiotic and antifungal properties. The release of allicin producesother sulfur derivatives, such as ajoene, allyl sulfides, diallylsulfides, allyl methyl disulfide, allyl methyl trisulfide, s-allylcysteine and diallyl trisulfide. Allicin pronouncedly inhibits thesecretion of various cytokines (IL-1b, IL-8, IP-10 and MIG) fromepithelial cells, suppressing the expression of interleukin 8 (1-8) andinterleukin 1b (IL-1b) mRN and is therefore considered to be effectivein attenuating intestinal inflammation. It is believed that low doses ofgarlic oil suppress NOS (inducible Notric Oxide Synthase) activity,ulceration and apoptosis of the intestinal mucosa. At high doses,however, garlic oil has shown a toxic effect, which is why it is deducedthat garlic is beneficial in moderate doses, but can be toxic in highdoses. Garlic extracts and garlic oil have also been found to bepowerfully anti-microbial against other GI bacteria such as Escherichiacoli, Shigella sp, Salmonella sp, and Proteus mirabills.

One aspect of the present invention is directed to the use of probioticsto modulate the human microbiota and promote health and preventantibiotic side effects. L. species are acid-resistant and commensal andtheir concentrations in the normal human stomach vary between 0 and10.sup.3 mL.sup.-1. They can survive in the stomach for periods of up to2 h. In various embodiments, fructo-oligosaccharides (FOS) andtrans-galacto-oligosaccharides (TOS), such as inulin, are used toselectively stimulate growth and activity of health-promoting bacteria.In this regard, dietary inulin fibers are used to stimulate Mg.sup.2+and Ca.sup.2+ absorption and are a potent stimulant of mineralabsorption, especially achieved by oligofructose-enriched inulin.Certain strains of gut bacteria have a preference for inulin fibers.Thus, one aspect of the present invention is to selectively advance thepopulation of such bacteria in a person's gut. It is known thatN-butryic acid increases Ca.sup.2+ and Mg.sup.2+ absorption. Thus,certain embodiments of the present invention are directed to theprovision of bacteria designed to produce n-butryic acid to treatPPI-induced Ca.sup.2+ disturbances. It is believed that dietary inulinstimulates intestinal Mg.sup.2+ absorption, and thus, other embodimentsof the present method include the provision of dietary inulin inaddition to the provision of the various bacteria strains as describedherein. One aspect of the present invention is therefore directed to theimpact of PPIs on Ca.sup.2+ homeostasis and provides a treatment forPPI-induced mineral disturbances. Dietary oligofructose enriched inulinfibers are believed to prevent omeprazole-induced reduction of Ca.sup.2+absorption and lead to improved intestinal Mg.sup.2+ absorption, thuspreventing PPI-induced mineral deficits in individuals.

In various embodiments, the present invention is directed to the use ofdietary inulin to counteract reduced intestinal Ca.sup.2+ absorptionupon PPI treatment. One aspect of the present invention is directed tothe local luminal acidification of the colon to enhance intestinalMg.sup.2+ absorption and by so doing, preventing PPIH. Other embodimentsare directed to the use of the fructan fiber inulin to reduce intestinalpH, such ingested inulin fibers being fermented in the large intestineby bifidogenic gut bacteria, resulting in short-chain fatty acids(SCFA), which in turn acidify the colon. Thus, one aspect of variousembodiments is directed to the stimulating action of SOFA on intestinalMg.sup.2+ absorption by reducing the luminal pH. Certain aspects aredirected to the enhancement of intestinal Mg.sup.2+ and Ca.sup.2+absorption in order to counteract omeprazole-induced defects in mineraluptake. Proton-pump inhibitor-induced hypomagnesemia (PPIH) is the mostrecognized side effect of proton-pump inhibitors (PPIs). Additionally,PPIH is associated with hypocalcemia and hypokalemia. It is hypothesizedthat PPIs reduce epithelial proton secretion and thereby increase the pHin the colon, which may explain the reduced absorption of and Mg.sup.2+and Ca.sup.2+. Fermentation of dietary oligofructose-enriched inulinfibers by the microflora leads to acidification of the intestinal lumenand by this enhances mineral uptake. One aspect of the present inventionis therefore directed to the improvement of mineral absorption byapplication of dietary inulin to counteract PPIH.

In various embodiments of the present invention, candidate probioticstrains are isolated from fecal samples, especially after enrichmentwith a prebiotic application. As described in other applicationsincorporated herein, the use of particular fecal samples from healthyAmish individuals is employed to combat GERD. Moreover, one strategy forenhancing the establishment of probiotic bacteria in the humanintestinal tract is via the parallel administration of a prebiotic. Invivo selection (IVS) may be employed to isolate candidate probioticstrains from fecal samples following enrichment with a prebiotic. Forexample, isolated bifidobacteria from human subjects who consumedincreasing doses of galactooligosaccharides (GOS) revealed an 8-foldenrichment in Bifidobacterium adolescentis strain IVS-1. It is believedthat such selected strains are able to outcompete residentBifidobacterium populations. One aspect of the present invention,especially employing one or more of the modified bacteria as describedherein, is to substantially enhance the establishment andcompetitiveness of one or more putative probiotic strains in anindividual's gastrointestinal tract to combat GERD.

One aspect of the present invention is directed to the provision ofpharmaceuticals based on an individual's own microbiome. Thus, incertain embodiments, isolation of particular bacteria from anindividual's stool is employed and CRISPR-Cas and/or Cpf1 systems arethen used to modify such bacteria in various beneficial ways, asdescribed herein. The reintroduction of such modified bacteria into theperson's gut (e.g. via fecal transplantation) provides a way toselectively and competitively compete with other undesired bacteria inthe person's gut. Preferably, the resident populations of gut microbesare reduced substantially before the reintroduction of the modifiedbacteria, thus providing a better chance and opportunity for theestablishment of a population of preferred bacteria, as modified via theCRISPR-Cas systems, as described herein.

The growth of microbiota communities is under control of distinctsubfamilies of host genes encoding antimicrobial peptides (AMPs). Whenbacteria colonize a given human habitat, the expression of AMPs,including .alpha. and .beta. defensins and cathelicidins, is upregulatedin order to limit the spreading of bacteria. The equilibrium between theimmune system and immunoregulatory functions of bacteria appears to be adelicate balance in which the loss of a specific species can lead to anoverreaction or suppression of the innate immune system. The maintenanceof a stable, fermentative gut microbiota requires diets rich in wholeplant foods particularly high in dietary fibers and polyphenols.Individuals colonized by bacteria of the genera Faecalibacterium,Bifidobacterium, LactoBacillus, Coprococcus, and Methanobrevibacter havesignificantly less of a tendency to develop obesity-related diseaseslike type-2-diabetes and ischemic cardiovascular disorders. Thesespecies are characterized by high production of lactate, propionate andbutyrate as well as higher hydrogen production rates, which are known toinhibit biofilm formation and activity of pathogens. Thus, in variousembodiments of the present invention, these bacterial species areselected and administered to an individual in preferred ratios thatreflect those of healthy individuals so as to attain the general balanceof bacterial populations in a person's gut. Moreover, preferablybacteria are selected that are effective in inhibiting biofilm formationand in particular, those that demonstrate a high production of lactate,propionate, butyrate and hydrogen. CRISPR-Cas and/or Cpf1 may beemployed to provide such characteristics to the selected bacterialspecies in this regard.

CDT (Clostridium difficile transferase) is a binary, actinADP-ribosylating toxin frequently associated with hypervirulent strainsof the human enteric pathogen C. difficile, the most serious cause ofantibiotic-associated diarrhea and pseudomembranous colitis. CDT leadsto the collapse of the actin cytoskeleton and, eventually, to celldeath. The lipolysis-stimulated lipoprotein receptor (LSR) is the hostcell receptor for CDT. By applying the CRISPR-Cas technology tointerfere with the binding component of CDT, preferably by impactingamino acids 757 to 866 of CDT, one is able to interfere with the bindingof CDT to the LSR. Thus, interfering with the interaction between CDTand its receptor LSR, is one way to provide an anti-toxin strategy forpreventing cell entry of the toxin. Use of the active expression ofCRISPR arrays in C. difficile strains is therefore one way in which tocounter Clostridium difficile nosocomial infections associated withantibiotic therapies. One aspect of certain embodiments is directed tomodifying the site where the bacterium Clostridium difficile's binarytoxin binds to intestinal cells' LSR (lipolysis-stimulated lipoproteinreceptor) protein and triggers a mechanism that results in the invasionof the host cells by the bacteria. Clostridium difficile produces thebinary, actin ADP-ribosylating toxin CDT (Clostridium difficiletransferase). While CDT can lead to death of the host cells throughcollapse of the actin cytoskeleton, low doses of CDT result in theformation of microtubule-based protrusions on the cell surface thatincrease the adherence and colonization of C. difficile. Thus, oneaspect of certain embodiments relates to the interference with theadherence characteristics of this bacteria by reducing the amount of CDTproduced by bacterial cells, thus providing a population of C. difficilethat do not pose the problems of wild type strains. One aspect ofcertain embodiments involves blocking certain areas in the toxin and thereceptor in order to prevent the Clostridium difficile transferase toxinfrom entering the host cell.

Other embodiments are directed to a system and method for reducing thelikelihood of GERD and includes the modification of an individual's gutmicrobes in a manner that reduces, if not eliminates, the symptoms ofGERD. Certain embodiments employ CRISPR-Cas or Cpf1 systems to render H.Pylori more susceptible to certain drugs, including antibiotics, thusaddressing the antibiotic resistance otherwise experienced by treatingH. Pylori with antibiotics.

Still other aspects of the present invention are directed to treatingaging in a fashion such that individuals do not suffer from the variousforms of cancer that invariably increase as an individual ages.Extending healthy life by slowing ageing is the most efficient way tocombat fatal and disabling pathologies, such as cancer, that plague theelderly human population. Thus, one aspect of the present invention isdirected not to overcoming age-associated pathologies one-by-one, butrather, to prevent or delay age-related pathologies in general, thusmore effectively addressing the commonplace chronic disordersexperienced by the elderly. The present invention therefore represents aparadigm shift in the current public health strategy, which is targetedto the prevention of particular disorders, which even if successful,leaves an individual susceptible to other comorbidities that inevitablysubstitute for the pathology being treated. In various embodiments ofthe present invention, a method and system is provided that treatsage-related disease by increasing the healthspan of a human individual.In various embodiments, rapamycin (sirolimus) is administered to anindividual via a person's microbiome by employing microbes, and inparticular bacteria modified to produce rapamycin and preferably otheranti-aging agents, to achieve this objective.

As described in more detail herein, one aspect of the present inventioninvolves the use of a natural small molecule derived from tomato plants,tomatidine, which is believed to cause cell growth, especially inskeletal muscle tissue. Tomatidine is an inhibitor of muscle atrophy andthus has a use as a therapeutic agent for skeletal muscle atrophy.Tomatidine is a steroidal alkaloid and the aglycone of alpha-tomatine,an abundant glycoalkaloid in tomato plants that mediates plant defenseagainst fungi, bacteria, viruses and predatory insects. When consumed byanimals, alpha-tomatine is hydrolyzed by stomach acid and intestinalbacteria to tomatidine, which is absorbed by the gut. Tomatidine isbelieved to have an anti-atrophic (anabolic) effect in skeletal muscleand possesses anti-hyperlipidemic and anti-atherosclerotic effectswithout evidence of toxicity. Tomatidine is significantly more potentthan ursolic acid in building muscle tissue and has a differentmechanism of action.

The tomato belongs to the Solanaceae family that includes more than3,000 species. Tomato fruit consumption has been associated with areduced risk of inflammatory processes, cancer, and chronicnoncommunicable diseases (CNCD) including cardiovascular diseases (CVD)such as coronary heart disease, hypertension, diabetes, and obesity.Tomatidine is found in certain plants at certain developmental stages,such as in green (but not ripened red) tomatoes. One aspect of thepresent invention is directed to the provision to individuals in needthereof with bacteria that have been modified to produce effectiveamounts of tomatidine to address the muscle atrophy associated withvarious cancers. In one embodiment, DNA encoding tomatidine or itsanalogs is inserted into the genome of one or more bacterial species byemploying CRISPR-Cas or CPf1 systems, such that an individual can orallytake a pill containing such modified bacteria (preferably bacteria ofthe same species as presently reside in the individual's gut microbiome)and in such a manner, administer tomatidine to the individual in amanner that does not require injections or the taking of traditionalpharmaceutical formulations containing tomatidine. In such a manner, theproduction by such bacteria inside the individual provides a morenatural way for tomatidine to be provided to those in need of itsextraordinary abilities to foster the retention of muscle mass in theindividual. The ability to further modify the populations of bacteriainside an individual via the use of particular antibiotics, for example,those that can target the modified species that produce tomatidine,provides a way to control the amount of tomatidine in the individual'sbody. Tomatidine in this instance, is but one of many examples of howthe personal microbiome of an individual can be amended, modified,enhanced and/or changed to adjust the levels and amounts of variouscompounds, drugs, molecules, etc. that are important in maintaining orrestoring health to an individual.

Yet another aspect of the present invention involves the treatment ofcancer cachexia, specifically including methods that use a natural smallmolecule derived from tomato plants, tomatidine, which is believed tocause cell growth, especially in skeletal muscle tissue. Tomatidine isan inhibitor of muscle atrophy and thus has a use as a therapeutic agentfor skeletal muscle atrophy. Tomatidine is a steroidal alkaloid and theaglycone of alpha-tomatine, an abundant glycoalkaloid in tomato plantsthat mediates plant defense against fungi, bacteria, viruses andpredatory insects. When consumed by animals, alpha-tomatine ishydrolyzed by stomach acid and intestinal bacteria to tomatidine, whichis absorbed by the gut. Tomatidine is believed to have an anti-atrophic(anabolic) effect in skeletal muscle and possesses anti-hyperlipidemicand anti-atherosclerotic effects without evidence of toxicity.Tomatidine is significantly more potent than ursolic acid in buildingmuscle tissue and has a different mechanism of action.

One aspect of the present invention is directed to the provision toindividuals in need thereof of bacteria that have been modified toproduce effective amounts of tomatidine to address the muscle atrophyassociated with various cancers and diseases. In one embodiment, DNAencoding tomatidine or its analogs is inserted into the genome of one ormore bacterial species by employing CRISPR-Cas or Cf11 systems, suchthat an individual can orally take a pill containing such modifiedbacteria (preferably bacteria of the same species as presently reside inthe individual's gut microbiome) and in such a manner, administertomatidine to the individual in a manner that does not requireinjections or the taking of traditional pharmaceutical formulationscontaining tomatidine. In such a manner, the production by such bacteriainside the individual provides a more natural way for tomatidine to beprovided to those in need of its extraordinary abilities to foster theretention of muscle mass in the individual. The ability to furthermodify the populations of bacteria inside an individual via the use ofparticular antibiotics, for example, those that can target the modifiedspecies that produce tomatidine, provides a way to control the amount oftomatidine in the individual's body. Tomatidine in this instance, is butone of many examples of how the personal microbiome of an individual canbe amended, modified, enhanced and/or changed to adjust the levels andamounts of various compounds, drugs, molecules, etc. that are importantin maintaining or restoring health to an individual.

In certain embodiments of the present invention, a method for treatingcancer cachexia involves the administering to the microbiome of asubject in need thereof of an effective amount of a bacterialcombination that expresses p53 protein and tomatidine, such cancer beingfor example, one of breast cancer, bladder cancer, kidney cancer, orcolorectal cancer. In certain embodiments, the cancer is a metastaticcancer; and the microbiome is one or more of the gut microbiome, theoral microbiome or the skin microbiome. Other embodiments involvemucosally administering to the subject an effective amount of a bacteriathat has been modified to express one of tomatidine, statins and/or p53,with the bacteria selected from the group consisting of Streptococcus,Actinomyces, Veillonella, Fusobacterium, Porphromonas, Prevotella,Treponema, Neisseria, Haemophilus, Eubacteria, Lactobacterium,Capnocytophaga, Eikenella, Leptotrichia, Peptostreptococcus, BacillusCalmette Guerin, Staphylococcus, and Propionibacterium. Still otherembodiments include the provision of Streptomyces hygroscopicus in anamount effective to produce therapeutically effective amounts ofrapamycin to the subject. It should be appreciated that atherapeutically effective amount is preferably an amount sufficient toelicit any of the listed effects of natural tomatidine and p53, forexample, including, but not limited to, the power to treat cancercachexia in a fashion demonstrated by a result indicating that there isevidence of the maintenance of muscle mass in the individual treated. Incertain preferred embodiments, the mucosal administration is oraladministration and the subject individual maintains or increases musclemass. In most preferred embodiments, the bacterial composition has beenmodified via a CRISPR-Cas or Cf11 system to express tomatidine, and inother embodiments, produces both tomatidine and p53 protein. Otherembodiments include a bacterial composition that includes one of aChlamydia species, Shigella flexneri, Mycoplasma bacteria, and/or H.pylori.

The TP53 gene, which encodes the p53 protein, is the most frequenttarget for mutation in tumors. In response to a number of stressors, p53becomes activated to promote cell cycle arrest, apoptosis or senescencethereby suppressing tumor growth and also plays many additional rolesincluding regulating cellular metabolism. Unlike most tumor suppressorgenes, which are predominantly inactivated as a result of deletion ortruncation, the majority of mutations in TP53 are missense mutations. Incontrast to wild-type p53, which under unstressed conditions is a veryshort-lived protein, missense mutations lead to the production offull-length p53 protein with a prolonged half-life. While manytumor-derived mutant forms of p53 can exert a dominant-negative effecton the remaining wild-type allele, serving to abrogate the ability ofwild-type p53 to inhibit cellular transformation, the end result is thatthe wild-type version of p53 is lost and the mutant form is retained,with cancer and cachexia resulting. There is substantial evidence thatcertain mutants of p53 can exert oncogenic, or gain-of-function,activity independent of their effects on wild-type p53. There is a greatneed for methods of treating cancer having mutated p53 protein and TP53.Certain embodiments of the present invention address such need byproviding methods and systems whereby the oncogenic mutant p53populations are hindered via the provision of agents that block theiractivities, such as statins, while also providing a new source ofwild-type p53 produced by added bacteria to the individual. Enhancing anindividual's microbiome with microbes that are also able to producetomatidine and rapamycin is a further way to combat various diseaseconditions and to restore health to the individual.

Over 50 percent of human tumors contain mutations in the gene encodingp53, a protein that plays a significant role in early carcinogenesis.Wild-type p53 is a tumor suppressor, but mutant p53 has been shown toexhibit gain-of-function properties as well, causing cancer formation byenabling normally suppressed pathways. One such oncogenic pathwayactivated by this mutation is the sterol biosynthesis pathway.3-Hydroxy-3-methylgutaryl CoA reductase inhibitors (statins) have proventherapeutic and demonstrate preventative effects in cardiovasculardiseases. Inhibition of this pathway using HMG-CoA Reductase inhibitors,commonly known as statins, forms various aspects of the presentinvention and provides an entirely new treatment against mutant p53expressing cancers, especially with respect to the delivery of statinsbeing via microbes administered to individuals. Statins are among themost carefully studied class of drugs in use and are well tolerated withan excellent safety profile. Statins are already FDA approved and widelyused for other indications. Statins also have other beneficialanti-inflammatory and immune-modulatory effects.

Various aspects of the present invention involve the confluence of somehistorically important developments in the biological sciences,including the recognition of the importance of the microbiome, theadvent of CRISPR-Cas systems to manipulate various genes and organisms,and the ability to better control the “holy grail” of cancer therapy,namely the use of competently folded p53 protein and the reduction ofthe actions of mutant p53 on the progression of cancer. With the furtheracknowledgement of tomatidine as one of the natural plant-derived agentsthat can have a positive effect on cachexia, especially that associatedwith cancer, as well as the beneficial effects from the administrationof rapamycin via an individual's microbiome, there is presently a newand extraordinary opportunity to advance the long sought but heretoforeunfulfilled prospects of effectively treating cancer so as tosubstantially extend the quality and quantity of life for thosesuffering therefrom.

Still further aspects of the present invention are directed to theappreciation that cancer and aging appear to be related in many ways, asthe ability of cancer to be immortal is a characteristic that ifunderstood, could be applied to the aging of cells, thus providing anability to combat age related diseases. By understanding how p53 can beadministered to cancer tissues to halt cancer progression, while at thesame time, stopping the ability of mutated p53 to promote cancer growth,a large step in providing a treatment for cancer is achieved. Thefurther provision of modified microbes administered to an individual'smicrobiome so as to produce effective amounts of particular agents, suchas tomatidine (to forestall muscle atrophy), rapamycin (to address agingissues), etc., is a further benefit that may be achieved, especiallygiven the CRISPR-Cas technologies that easily provide an unprecedentedability to modify microbes in this regard—and without having togenetically engineer human cells in the process of such treatments.

Still other embodiments of the present invention are directed to theemployment of anaerobic bacteria to address cancer growth as thebiological environment of cancerous tumors and anaerobic bacteria aresimilar in several respects. Abnormal blood vessels and hypoxic andnecrotic regions are universal features of solid tumors. These hypoxicand anoxicmicroenvironments may be targeted by obligatory or facultativeanaerobic bacteria, such as Bifidobacterium, Salmonella, Escherichia,lostridium and Listeria, as well as certain bacteria believed to beinvolved in the progression of Alzheimer's Disease, namely, spirochetes.Some of these bacteria accumulate and actively proliferate withintumors, resulting in much higher increases in the number of bacterialcells in tumor tissues relative to those in normal organ cells andtissues, such as liver and spleen. The use of attenuated bacterialstrains to suppress tumor growth forms one aspect of the presentinvention, and in particular, use of BCG that has been further modifiedto express particular agents, such as tomatidine, p53, statins,rapamycin, etc. whether in separate bacterial cells or the samebacterial cell, comprises various aspects of the present invention. Suchmodified bacteria may act by directly suppressing tumor growth directlyand/or by activating host immunity, but are effective in achievingimproved therapeutic effects. One advantage of various embodiments ofthe present invention is that instead of having to inject bacteria intoa patient's tumor, the administration of agents is achieved preferablyvia the more organic administration of agents via the bacterial and hostcell interactions. Various of the embodiments encompassed herein relyupon approaches that stimulate inflammation, and thus trigger anantitumor immune response by the individual.

Other aspects of certain embodiments of the present invention aredirected towards the administration of quercetin, a polyphenol abundantin plants, preferably as it is or in the form of a glycoside. Quercetinmay be found in various plants including citrus fruits, onion,buckwheat, and Sophora and it is known that it has a wide variety ofphysiological functions, such as platelet aggregation/adhesioninhibition activities, and vasodilatory activity. In various embodimentsof the present invention, quercetin is expressed by cells of anindividual's microbiome to elicit anticancer activity and to conferother health benefits. The administration of certain modified bacteriato an individual in various embodiments effectively creates microbialcell factories that, through metabolic engineering of heterologousbiosynthetic pathways, turns microbes into a cost effective and moreorganic way to both produce and administer agents to desired tissues ina person suffering from cancer and other diseases. Using CRISPR-Castechniques especially, and in view of the guidance provided herein, oneof ordinary skill in the art will be able to excise desired genes fromplants, bacteria and fungi and have them expressed by microbes that canbe administered to an individual's microbiome such that health can berestored to the individual.

In addition, treatments for various types of cancer are desired thatrelate to the production of competently folded p53 tumor support factor.There has been a long felt but unmet need for a way to inexpensivelyadminister desired amounts of p53 protein to an individual in needthereof. The present invention in several of its aspects addresses thisconcern, for example, by the expression of p53 by human microbiomebacteria, by the administration of BCG cells transformed to express p53(as well as statins, rapamycin, tomatidine, quercetin, neoalbaconol, aphosphatase and tensin homolog (PTEN), etc.

Thus, in particular embodiments, the present invention is specificallydirected to a method of treating cancer cachexia in a subject in need ofsuch treatment by administering a therapeutically effective amount of acomposition comprising Bacillus calmette-guerin that is adapted toproduce tomatidine, wherein the cancer is bladder cancer oralternatively colorectal cancer. The Bacillus calmette-guerin ispreferably modified via a CRISPR-Cas system to produce tomatidine, iseven more preferably adapted to also produce rapamycin, and even morepreferably adapted to further produce a statin, such as one or more ofMevacor™, Pravachol™, simvastatin, atorvastatin, fluvastatin,lovastatin, pravastatin and rosuvastatin. In still other embodiments,the Bacillus calmette-guerin is adapted to produce neoalbaconol, andeven more preferably, it is further adapted to produce p53. Otherembodiments are similar to that described above, but the Bacilluscalmette-guerin is specifically adapted to produce tomatidine and thecancer is colorectal cancer. One will appreciate that similar preferredembodiments include Bacillus calmette-guerin being further adapted toproduce one or more of rapamycin, a statin, a phosphatase and tensinhomolog (PTEN), p53 and/or neoalbaconol.

Treatments for various types of cancer are desired that relate to theproduction of competently folded p53 tumor support factor. There hasbeen a long felt but unmet need for a way to inexpensively administerdesired amounts of p53 protein to an individual in need thereof. Thepresent invention in several of its aspects addresses this concern, forexample, by the expression of p53 by human microbiome bacteria. Incertain embodiments of the present invention, a method for treatingcancer cachexia involves the administering to the microbiome of asubject in need thereof an effective amount of a bacterial combinationthat expresses p53 protein and tomatidine, such cancer being forexample, one of breast cancer, bladder cancer, kidney cancer, orcolorectal cancer. In certain embodiments, the cancer is a metastaticcancer; and the microbiome is one or more of the gut microbiome, theoral microbiome or the skin microbiome. Other embodiments involvemucosally administering to the subject an effective amount of a bacteriathat has been modified to express one of tomatidine and p53, with thebacteria selected from the group consisting of—Streptococcus,Actinomyces, Veillonella, Fusobacterium, Porphromonas, Prevotella,Treponema, Neisseria, Haemophilus, Eubacteria, Lactobacterium,Capnocytophaga, Eikenella, Leptotrichia, Peptostreptococcus,Staphylococcus, Streptococcus thermophilus and Propionibacterium. Stillother embodiments include the provision of Streptomyces hygroscopicus inan amount effective to produce therapeutically effective amounts ofrapamycin to the subject. It should be appreciated that atherapeutically effective amount is preferably an amount sufficient toelicit any of the listed effects of natural tomatidine and p53, forexample, including, but not limited to, the power to treat cancercachexia in a fashion demonstrated by result indicating the maintenanceof muscle mass in the individual treated. In preferred embodiments, themucosal administration is oral administration and the subject individualmaintains or increases muscle mass. In most preferred embodiments, thebacterial composition has been modified via a CRISPR-Cas or CPf1 systemto express tomatidine, and in other embodiments, produces bothtomatidine and p53 protein. Other embodiments include a bacterialcomposition that includes one of a Chlamydia species, or Shigellaflexneri, Mycoplasma bacteria, and H. pylori.

With respect to combating aging, particular agents and combinationsthereof are employed in various embodiments of the present inventionsuch that the incidence of cancer is lessened due to the avoidance ofaging. Certain embodiments involve the administration of microbes to anindividual where such microbes have been modified to produce desiredlevels of such agents/combinations. Since signaling pathways related tothe aging of Caenorhabditis elegans (C. elegans), fruit flies and miceare evolutionarily conserved, compounds and agents extending lifespan insuch organisms are believed to be useful in treating age-relateddiseases in humans. Natural products are preferred as they have aspecial resource advantage with few side effects. For example, microbescan be introduced to an individual's microbiome so that that they mayproduce one or more of such desired natural product agents by anindividual's microbiome. One such agent is rapamycin (aka sirolimus),which is a macrolide produced by the bacterium Streptomyceshygroscopicus. Sirolimus is currently used as an immunosuppressant andis most often used to prevent rejection of transplanted organs.Sirolimus has two approved indications—renal transplantation andlymphangioleiomyomatosis and has also been shown to be potentiallyeffective in treating Tuberous Sclerosis Complex (TSC)-associatedseizures, skin disease, brain lesions, pulmonary lesions, and renallesions. Administration of therapeutically effective amounts ofrapamycin via an individual's microbiome (whether oral vaginal, skin,gut, bladder, etc.) forms one aspect of various embodiments of thepresent invention. Such employment of an anti-aging medicine likerapamycin is believed to be one of the most effective ways to combatvarious age-associated diseases of aging people. Rapamycin is but oneexample of the many compounds with anti-aging activity that have beenand will be discovered. Delivery of such agents by employing anindividual's microbiome is believed to be an effect and person-specificway to account for the vast diversity of individual's biologicalsystems, including the acknowledged disparity and array of microbiomecompositions.

Rapamycin is an inhibitor of mTOR complex (mammalian target ofrapamycin) which is a serine threonine kinase and a master regulator ofprotein synthesis, cell growth, and cell metabolism. Excessive mTORC1activity has been implicated in multiple disease conditions, as well asvarious cancers, inflammatory bowel disease, inflammatory skin diseasesand neurodegenerative diseases. In various embodiments, rapamycin isemployed, especially when produced by microbes in an individual'smicrobiome, to treat or prevent disease conditions by inhibiting themTORC1 pathway.

In certain embodiments, and while not bound by theory, it is believedthat tomatidine increases the ability of an individual to maintainmuscle mass, while rapamycin, as an inhibitor of mTOR, which increasesthe production of muscle proteins, reduces the growth of muscles. It isbelieved that these two agents may play parallel but separate roles inmuscle atrophy, and thus, the use of both of these agents to addresscancer, cachexia and aging is one particular aspect of the presentinvention.

In the treatment of particular cancers, the employment of rapamycin toinhibit cell growth, especially muscle growth, may in various instancesbe desired. At the same time, retention of muscle mass may be importantfor an individual to withstand the rigors of various cancer treatments.By administering tomatidine to an individual to maintain desired musclemass, while also co-administering rapamycin to such individual toinhibit the growth of certain cells, especially cancer cells, one isable to achieve the seemingly converse objectives of maintaining musclemass so as to preserve the health of an individual, while simultaneouslydefeating the undesired growth of cancer cells by the administration ofeffective amount of rapamycin to inhibit such undesired growth.

As described herein, p53 has apoptotic characteristics and effectivelykeeps cancer growth in check by preventing cells from growinguncontrollably. In a somewhat similar manner, rapamycin also may beemployed to regulate growth (e.g. by inhibiting growth of particularcells). Thus, the combination of rapamycin and p53 expression via cellsof an individuals microbiome provides two agents that are criticalcomponents in cell growth and apoptosis events at the core of cancertreatments. Effective administration of cells (or microbes) of aperson's microbiome via the purposeful administration of such cells(which have been modified, preferably via CRISPR-Cas systems) to expresstherapeutically effective amounts of either or both p53 and rapamycin,is believed to provide an effective treatment for various cancerousconditions.

It is noted that caloric restriction would seem to negatively affect thegrowth of an organism and detrimentally affect the protein expressionthat would otherwise ensue in a well-fed individual. Rapamycin is agrowth inhibitor, and thus, one would similarly conclude that theemployment of such an agent would reduce the expression of proteins,such as those that are employed to build muscle. Moreover, p53 is anagent that generates cell death via apoptosis, and thus, would be viewedas an anti-growth factor in terms of cell survival. And yet all three ofthese agents are considered instrumental in both the aging process aswell as in cancer. As cancer and aging are linked on certain levels, sotoo are the above referenced agents. The employment of these agents,especially by their purposeful expression via an individual's modifiedmicrobiome, provides a unique and effective way in which to combat bothcancer and aging. Various embodiments of the present invention aredirected to a method for treating, inhibiting, or reducing aging, anage-related symptom, and/or an age-related disease (e.g. cancer) in asubject which comprises administering to the subject a therapeuticallyeffective amount of a compound that has anti-aging characteristics, withadministration being preferably via an individual's own microbiome.Included, but not limited to such a list of compounds is rapamycin, p53and tomatidine.

The administration of such compounds/agents via an individual'smicrobiome is believed to positively affect the extending of thelifespan of an individual, and especially effective in delaying theonset of age-related diseases and conditions, such as cancer, thusextending the healthspan of the individual from what it otherwise wouldhave been if such administration was not performed. The particulareffective amount of such agents/compounds, such as rapamycin (includinganalog or derivatives thereof) depend upon the disease to be treated,the length of duration desired and the particular characteristics of theindividual's microbiome—and which of the one or more various microbiomesof the person may be the source of the administration. In certainembodiments where the agent/compound comprises rapamycin or an analogthereof, administration of rapamycin may be performed to affect about0.001 mg to 30 mg total per day as an effective dose, preferably atleast about 0.1 mg per day, with a preferred blood level of rapamycin inthe subject being about 0.5 ng per mL whole blood after administrationof the composition after a 24 hour period. By administering antibioticsthat target the particular microbes that produce such agents/compounds(e.g. rapamycin) one can address overproduction by such microbes bykilling the microbes producing such agents. Various other embodimentsare directed to the skin microbiome of a person so as to addressdiseases of the skin, including but not limited to skin cancer. Thelactic acid bacteria Streptococcus thermophilus has been found toincrease ceramide production in the skin. Ceramides are known to play anessential role in structuring and maintaining the water permeabilitybarrier function of skin. With aging, the total ceramide content ofskin, along with the skin's ability to function as a barrier, decreases.Certain embodiments are directed to ceramide supplementation via asubject's microbiome to improve skin barrier function.

Certain embodiments are directed to a method of treating bladder cancerin a subject in need of such treatment, such method comprisingadministering to a microbiome of a subject with bladder cancer aneffective amount of a bacterial composition comprising Bacilluscalmette-guerin, with the bacterial composition adapted to producetomatidine and rapamycin. Preferably, the bacterial compositioncomprises bacteria modified via a clustered regularly interspaced shortpalindromic repeats (CRISPR) CRISPR associated protein (Cas) system toexpress one or both of tomatidine and rapamycin. Certain embodiments arefocused on treating metastatic bladder cancer. The microbiome employedmay be the gut, oral, bladder or skin microbiome. Certain embodimentsfurther include employing a microbe selected from the group consistingof Streptococcus, Actinomyces, Veillonella, Fusobacterium, Porphromonas,Prevotella, Treponema, Nisseria, Haemophilis, Eubacteria,Lactobacterium, Capnocytophaga, Eikenella, Leptotrichia,Peptostreptococcus, Staphylococcus, and Propionibacterium. One preferredembodiment involves administering a bacterial composition to the subjectso that at least 0.1 mg of rapamycin is provided to the subject eachday. Preferably, the bacterial composition is modified via a CRISPR-Cassystem to express one of rapamycin and/or tomatidine, with preferredbacterial compositions including one of a Chlamydia, Shigella flexneri,Mycoplasma bacteria, and H. pylori. In other preferred embodiments, themethod comprises administering to a microbiome of a subject with bladdercancer an effective amount of a bacterial composition comprising abacteria that has been modified to express a therapeutically effectiveamount of tomatidine and rapamycin, with the bacteria selected from thegroup consisting of Streptococcus, Actinomyces, Veillonella,Fusobacterium, Porphromonas, Prevotella, Treponema, Nisseria,Haemophilis, Eubacteria, Lactobacterium, Capnocytophaga, Eikenella,Leptotrichia, Peptostreptococcus, Staphylococcus, Propionibacterium,Chlamydia, Shigella flexneri, Mycoplasma bacteria, H. pylori, andStreptomyces hygroscopicus. The bacteria employed may be of a speciesfound in the subject's gut microbiome and may further have been modifiedusing a CRISPR-Cas system to produce one of tomatidine or rapamycin. Atherapeutically effective amount of a bacterial composition may alsoinclude Streptomyces hygroscopicus in an amount effective to provide atherapeutically effective amount of rapamycin to the subject. Inparticular embodiments, especially directed to addressing bladdercancer, the bacterial composition comprises Bacillus calmette-guerin,and even more preferably, where the Bacillus calmette-guerin alsoproduces at least one of p53, rapamycin or tomatidine, and especiallywhere the method maintains or increases the muscle mass of the subject.As described in more detail in the detailed description of variousembodiments, still other agents, such as methylene blue, metformin,resveratrol (3,4′,5-trihydroxystilbene; C.sub.14H.sub.12O.sub.3), p53protein, spermidine, diallyl trisulfide, apigenin, cyclopamine,sulforaphane, curcumin and glucosamine are employed via the productionby microbes of an individual's microbiome to achieve the objective ofdelaying aging, and thus, in delaying and treating the onset of cancers.

One aspect of the present invention is directed to the use of humanspecific species of bacteria that are then modified to enhance one ormore characteristics deemed beneficial to the skin microbiome and healthof the individual, including treating skin cancers. Many embodimentsemploy bacteria that have been modified via a CRISPR-Cas9 and/or Cpf1system to either repress the expression of a particular protein orlipid, or to increase the production of beneficial microbial secretions.Clustered Regularly Interspaced Short Palindromic Repeats fromPrevotella and Francisella 1 or CRISPR/Cpf1 is a DNA-editing technologyanalogous to the CRISPR/Cas9 system. One objective is to avoid modifyingan individual's human genome, but instead, to significantly affect thehealth of humans by employing modifications to the skin microbiome. Useof human specific strains of bacteria, whether they are commensal orpathogenic, including bacteria that are modified to alter their nativepathogenicity, is one preferred aspect of many embodiments of thepresent invention.

Certain aspects of the present invention are directed to a method foraltering the microbiome of an individual's skin by administering to aregion of the skin of an individual an effective amount of a bacterialformulation. In one preferred embodiment, the individual is a newbornand the step of administering is performed within the first 6 hours ofthe newborn's birth. Such a bacterial formulation may be a lotion,ointment or gel adapted to be rubbed onto the newborn's skin. Thebacteria included in the bacterial formulation may vary to addressparticular concerns or diseases. For example, the bacterial formulationmay include bacteria selected from the group consisting of Nitrosomonaseutropha and Propionibacterium. More particularly, the equilibrium of abacterial population of the region of the skin of the individual ismodified to increase the number of Propionibacterium bacteria and todecrease the number of Staphylococcus bacteria on the individual's skinin such region. In other embodiments, the bacterial formulation includesthe bacteria Staphylococcus aureus that has been modified by employing aCRISPR-Cas or Cpf1 system to interfere with S. aureus virulenceregulation involving the Agr quorum-sensing signaling molecule. Inseveral embodiments, the bacterial formulation comprises a bacteria thathas a tropism specific for the human species. In others, the bacterialformulation comprises at least two of the bacteria selected from thegroup consisting of: Prevotella; LactoBacillus johnsonii; Bacteroidesfragilis, LactoBacillus ruminus and L. infantitis. In certainembodiments the bacteria is an ammonia oxidizing bacteria. In otherembodiments, the region of the skin to which the bacterial formulationis applied is the scalp. In various embodiments, rather than using awild-type bacteria, the bacteria employed is one that has been modifiedby CRISPR-Cas or CRISPR-Cpf1 to delete a functional virulence factorfrom the bacteria. In particular embodiments, the method includesadministering to the skin a bacteria that produces tomatidine. Inothers, the bacteria produces p53. Thus, in some embodiments, the methodinvolves use of bacteria wherein a CRISPR-Cas or CRISPR-Cpf1 system isemployed to insert a gene for the production of tomatidine and/or p53into at least one of the bacteria in the bacterial formulation. Inothers, a CRISPR-Cas or CRISPR-Cpf1 system is employed to insert one ormore genes into the bacteria comprising the bacterial formulation tofacilitate the oxidizing of ammonia by the bacteria. To further enhancethe ability of desired bacteria to be maintained on the skin of anindividual, certain methods further comprise administering to theindividual's skin a prebiotic that comprises a nutrient source for thebacteria that is assimilated by the bacteria, and preferably one that isnot digestible by the individual. In particular embodiments, the methodfurther includes administering to the skin an extract derived from ahelminth selected from the group consisting of Capillaria hepatica,Dicrocoelium dendriticum, Ascaris lumbricoides, Enterobius vermicularis,Trichuris trichiura, Ancylostoma duodenale, Necator americanus,Strongyloides stercoralis, Haemonchus contortus, and Trichinellaspiralis. In still others, the bacterial formulation includes at leastone arabinogalactan. Yet others include at least one of the following:L. infantitis, and L. johnsonii. In a particular embodiment, thebacterial formulation includes at least one bacteria modified via aCRISPR-Cas system to express a gene encoding interferon regulatoryfactor 4.

In particular embodiments, in view of the tropism demonstrated by S.pyogenes for humans, and the recognition that such bacterial species isfound in both the oral and skin microbiome of humans, S. pyogenes is apreferred bacterial species to employ in various embodiments of thepresent invention.

In still other embodiments, the focus is on interspecies interactionswithin mixed microbial communities, with the objective being to modifycompetitive relationships involving nonbiocidal biosurfactants, enzymes,and metabolites produced by bacteria and other microorganisms in amanner such that selection of particular bacterial species can beemployed to inhibit initial adhesion, trigger matrix degradation,encourage jamming of cell-cell communications, and induce biofilmdispersion. Nonbiocidal molecules are thus employed to modifycompetitive interactions within biofilms in a manner that promotes theoverall health of an individual's microbiome, especially on the skin.

In certain embodiments, a bacterial formulation is applied to newbornswithin a critical window of time after birth, preferably within thefirst 24 hours of the newborn's birth, more preferably within 6 hours oftheir birth, even more preferably within 3 hours of birth, and mostpreferably within an hour after their birth. The administration can beby several methods, but preferably is a lotion, ointment or gel that isrubbed onto the newborn's skin, preferably all over his/her entire body.A spray or mist can also be applied that contains the bacterial andmicrobe formulations as set forth herein. While not bound by theory, thecritical window to apply to the newborn's skin the referencedformulations, e.g. microbial mixtures of bacteria beneficial intriggering immune system development, is within a relatively short timeperiod and is necessary to establish immune tolerance to a variety ofcommensal microbes. The way and content of microbes presented at a timein which a newborn has his/her skin colonized establishes immunetolerance to particular commensal microbes. The influx of highlyactivated T cells into neonatal skin is believed to occur in suchcritical window. So a mother of a newborn has a choice: to simply relyupon chance as to what particular microbes might be present during thiscritical window of the newborn's establishing immune tolerance toparticular bacteria and other microbes; or to provide the newborn with aselected formulation containing predetermined microbes such that thenewborn's developing immune system can properly react to the microbes inthe predetermined formulation, and thus provide the newborn with theopportunity to develop a more expansive immune tolerance profile. It isbelieved that the mechanism that promotes tolerance is tissue specific,and thus, the skin and the gut may have different ways by which theymediate tolerance to commensal microbes. To establish a healthy statusof a newborn's skin as it relates to commensal microbes on its skin, theparticular type of microbes, including bacteria, brought into contactwith his/her skin is achieved in a certain time period after birth (e.g.within 1 to 24 hours after birth) so that the developing immune systemof the infant establishes tolerance to such microbes, thus avoidingallergies, autoimmune diseases and other related diseases, as well aschronic inflammation of the skin.

In certain embodiments of the present invention, the skin microbiome isenhanced via providing microbes able to metabolize lipids, proteins andcarbohydrates, and thus, produce acid that aids in maintaining theso-called “acid mantel” of the skin. In preferred embodiments thebacteria that is modified has a very narrow host tropism, such that thebacteria are specific for the human species and thus, their modificationposes little if any risk to other animals or organisms.

Other embodiments are directed to combating infections of a person'sskin by the bacteria Staphylococcus aureus. Staphylococcus aureus is acommensal and pathogen of both humans and cattle. In certain embodimentsthe accessory gene regulator (Agr) system and the virulence regulationof S. aureus pathogenesis is modified to delete or to at least reducethe virulence of the bacteria. In such a way, the present inventionprovides a way to effectively combat S. aureus infections. In variousembodiments of the present invention, CRISPR-Cas9 and/or Cpf1 systemsare employed to render ineffective virulence factors of such bacteriainvolved with the establishment and propagation of infection. Severalmolecules have been found to interfere with S. aureus virulenceregulation, especially those targeting the Agr quorum-sensing signalingmolecule. By modification of this bacterial species using CRISPR-Casand/or Cpf1 it is possible to achieve broad-spectrum inhibitory effectson most S. aureus strains and Agr subtypes.

The tropism of individual bacteria for particular host tissues (e.g.,skin vs. respiratory tract vs. gastrointestinal tract) is determined bythe array of available adhesion-receptor pairs. In preferredembodiments, bacteria having substantial, if not entire, human hostspecificity are employed. For example, Salmonella enterica serovarTyphi, known to be the bacteria responsible for typhoid fever, alife-threatening human disease, demonstrates strict human hostspecificity. In certain embodiments, the virulence factors of suchbacteria are compromised by being modified via the CRISPR-Cas or Cpf1system to render the modified bacteria as non-pathogenic. Similarly, thebacteria Neisseria, the causative agent of gonorrhea, is a diseaserestricted to humans, and thus similar CRISPR-Cas and/or Cpf1 systemsmay be employed to reduce if not eliminate the virulence factors of suchbacteria. Likewise, Helicobacter pylori is known to be an etiologicagent of gastritis and peptic ulcer disease in humans. The ironacquisition system of H. pylori by the human lactoferrin receptor systemis believed to play a major role in the virulence of H. pyloriinfection. The CRISPR-Cas and/or Cpf1 systems may be employed to reduceif not eliminate the virulence factors of this bacteria. Yet anotherbacteria demonstrating human tropism is Haemophilus influenza, a Gramnegative species that requires heme and has exclusive human hostspecificity. In certain embodiments, the CRISPR-Cas and/or Cpf1 systemsmay be employed to reduce if not eliminate the virulence factors of suchbacteria. The distinction between throat and skin group A Streptococcushas become blurred and to date there have been few advances in treatmentof group A Streptococcus skin infections. Certain aspects of the presentinvention include the modification of skin group A Streptococcus toreduce the likelihood, if not prevent, related skin diseases, includingeczema, atopic dermatitis, acne, allergic inflammation, skinhypersensitivity, UV-induced skin damage, and skin cancer.

One particular aspect of certain embodiments of the present inventionrelates to the treatment of acne. Acne is the most common skin diseaseaccounting for a quarter of dermatologists' patient volume. Acne is achronic disease that can significantly impact an individual's quality oflife with social, psychological and emotional impairments. Thus, invarious embodiments, bacteria are selected that, once applied to anindividual's skin, is able to ameliorate acme. Such bacteria includepreferably ammonia oxidizing bacteria, preferably provided to a person'sskin in combination with a pharmaceutically acceptable excipient. Incertain embodiments, bacteria are employed to achieve topical nitricoxide release at or near the surface of the skin and addition of urea orammonium salts to the skin provides additional substrates that thesebacteria utilize to form nitrite. While not intending to be limitedthereby, such ammonia oxidizing bacteria may be selected from the groupconsisting of Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis,Nitrosolobus, Nitrosovibrio, and combinations thereof. In someinstances, the ammonia oxidizing bacteria is Nitrosomonas eutropha (N.eutropha). Such ammonia-oxidizing bacteria are employed to improve skinhealth and are able to convert ammonia to nitrite, an anti-microbialcompound, and nitric oxide. Various aspects of the present invention aredirected at restoring and maintaining the delicate balance of the skinmicrobiome.

The present invention in various embodiments is directed to a variety ofconsumer products including cosmetic products such as skin care products(bath preparations, skin washing and cleaning products, skin careproducts, eye cosmetics, lip care products, nail care products, intimatehygiene preparations, foot care), those with special effects(sunscreens, tanning agents, deodorants, anticholinergics, depilatories,shaving, fragrance), those for oral or dental hygiene and those for haircare (shampoos, conditioners, etc.)

One objective of the present invention is to achieve various health andcosmetic benefits by providing a healthy, balanced skin microbiome.Modified bacteria that are beneficial to the skin, especially thosemodified using CRISPR-Cas systems, are used to enhance the beneficialcharacteristics of skin microbiomes in a manner that purposefullyexposes skin to microbes, rather than the conventional use ofanti-bacterial agents to kill bacteria—including beneficial bacteria—ona person's skin. The adherence to the skin of problem flora, such aspathogenic bacteria and yeast, has been associated with numerousailments, including skin infections, diaper rash, urinary or vaginalinfections, and malodors. Use of the present invention addresses suchissues in a novel and non-obvious manner.

Other embodiments are directed to prebiotic agents for use on skin. Inpreferred embodiments, CRISPR-Cas and/or Cpf1 modified bacteria,especially those demonstrating total or substantial tropism for humans,are employed in one or more of the above referenced products, withcertain features, namely, virulence factors reduced if not eliminated.In such a manner, there is a competitive inhibition of undesiredbacteria with the modified bacteria as set forth herein. In certainembodiments, the cleansing of one's skin to effectively reduce by atleast about 50%, more preferably about 30%, and most preferably toreduce by at least about 25%, of native bacteria on an individual's skinportion to be addressed, is performed prior to purposefully contactingthe individual's skin with one or more bacteria, and in particular,bacterial species that have been modified via employment of a CRISPR-Casand/or Cpf1 system to reduce if not effectively compromise the virulencefactors of such bacteria, and more preferably a bacteria that has a hostspecificity exclusive to humans.

In one particular embodiment, bacteria are modified via a CRISPR-Cassystem to express a gene identified for grey hair—interferon regulatoryfactor 4 (IRF4). This gene is involved in regulating production andstorage of melanin, the pigment that determines hair, skin and eyecolor. Hair greying is caused by an absence of melanin in hair. Thus, onvarious embodiments, bacteria are modified to express IRF4 and topicalapplication of such bacteria to an individual's scalp provides for theprevention of hair turning grey as it otherwise would without suchapplication of such bacteria. In still other embodiments, bacteria aremodified to express levels of melanin to maintain hair color when suchmodified bacteria are contacted with the scalp of an individual.

Particular aspects of the present invention are directed to a method forreducing the likelihood of developing cancer in an individual humanbeing by providing in the gut of an individual a population ofbeneficial bacteria selected from the group consisting of LactoBacillusspecies. To maintain such bacteria, it is preferred that the individualbe administered at least 6 grams per day of fiber. Moreover, the numberand/or level of particular bacteria, namely, Roseburia andFaecalibacterium prausnitzii, are increased in the individual's gutmicrobiome. Then the individual is administered an immune checkpointinhibitor. It is believed that the checkpoint inhibitor's desiredfunction is enhanced due to the presence of the bacterial populationstated above. The immune checkpoint inhibitor may be selected from thegroup of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514,STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C,AUR-012 and STI-A1010. In certain embodiments, the bacteria have beenmodified by using a clustered regularly interspaced short palindromicrepeats (CRISPR) CRISPR associated protein (Cas) system or a CRISPR fromPrevotella and Francisella 1(Cpf1) system to remove a virulence factor.Any of the later CRISPR systems can also be employed for such purpose.Moreover, still other embodiments employ the provision to an individualhuman being's gut of a population of Akkermansia muciniphila bacteria.Similarly, administering at least 6 grams per day of fiber to theindividual maintains a therapeutically effective amount of theAkkermansia muciniphila bacteria in the gut, and thus, when theindividual human being is administered an immune checkpoint inhibitor,the function and positive results of such cancer treatment is enhanced.

Yet further embodiments include the increase in the levels of Roseburiain the gut of the individual human being. Other embodiments similarlyinvolve increasing the levels of Faecalibacterium prausnitzii in the gutof the individual human being. Still others involve increasing thelevels of bacterial species selected from the group consisting ofBifidobacterium, Prevotella, Lachnospira, and Shigella. It should beunderstood that in combating cancer, other embodiments involveadministering to an individual human being bacteria that have beenmodified using a CRISPR-Cas system to produce p53. Such bacteria may beselected from the following in certain preferred embodiments:Streptococcus, Actinomyces, Veillonella, Fusobacterium, Porphyromonas,Prevotella, Treponema, Neisseria Haemophilus, LactoBacillus,Capnocytophaga, Eikenella, Leptotrichia, Peptostreptococcus,Propionibacterium, Chlamydia, Shigella flexneri, Mycoplasma bacteria, H.pylori, and Streptomyces hygroscopicus.

One will appreciate that this Summary of the Invention is not intendedto be all encompassing and that the scope of the invention nor itsvarious embodiments, let alone the most important ones, are necessarilyencompassed by the above description. One of skill in the art willappreciate that the entire disclosure, as well as the incorporatedreferences, figures, etc. will provide a basis for the scope of thepresent invention as it may be claimed now and in future applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a picture of Faecalibacterium prausnitzii.

FIG. 2 is a picture of Akkermansia muciniphila.

FIG. 3 is a picture of Roseburia.

FIG. 4 is a picture of Clostridium.

FIG. 5 is a picture of Veillonella.

FIG. 6 is a picture of Prevotella.

FIG. 7 is a picture of Propionibacterium.

FIG. 8 is a picture of Pseudomonas aeuroginosa.

FIG. 9 is a picture of Klebsiella

FIG. 10 is a picture of Shignella.

FIG. 11 is a picture of Acinetobacter baumannii.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

“CRISPR” (Clustered Regularly Interspaced Short Palindromic Repeats)loci refers to certain genetic loci encoding components of DNA cleavagesystems, for example, used by bacterial and archaeal cells to destroyforeign DNA. A CRISPR locus can consist of a CRISPR array, comprisingshort direct repeats (CRISPR repeats) separated by short variable DNAsequences (called spacers), which can be flanked by diverse Cas(CRISPR-associated) genes. The CRISPR-Cas system, an example of apathway that was unknown to science prior to the DNA sequencing era, isnow understood to confer bacteria and archaea with acquired immunityagainst phage and viruses. Intensive research over the past decade hasuncovered the biochemistry of this system. CRISPR-Cas systems consist ofCas proteins, which are involved in acquisition, targeting and cleavageof foreign DNA or RNA, and a CRISPR array, which includes direct repeatsflanking short spacer sequences that guide Cas proteins to theirtargets. Class 2 CRISPR-Cas are streamlined versions in which a singleCas protein bound to RNA is responsible for binding to and cleavage of atargeted sequence. The programmable nature of these minimal systems hasfacilitated their use as a versatile technology that is revolutionizingthe field of genome manipulation.

As used herein, an “effector” or “effector protein” is a protein thatencompasses an activity including recognizing, binding to, and/orcleaving or nicking a polynucleotide target. An effector, or effectorprotein, may also be an endonuclease. The “effector complex” of a CRISPRsystem includes Cas proteins involved in crRNA and target recognitionand binding. Some of the component Cas proteins may additionallycomprise domains involved in target polynucleotide cleavage.

The term “Cas protein” refers to a polypeptide encoded by a Cas(CRISPR-associated) gene. A Cas protein includes proteins encoded by agene in a cas locus, and include adaptation molecules as well asinterference molecules. An interference molecule of a bacterial adaptiveimmunity complex includes endonucleases. A Cas endonuclease describedherein comprises one or more nuclease domains. A Cas endonucleaseincludes but is not limited to: the novel Cas-alpha protein disclosedherein, a Cas9 protein, a Cpf1 (Cas12) protein, a C2c1 protein, a C2c2protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, Cas10, orcombinations or complexes of these. A Cas protein may be a “Casendonuclease” or “Cas effector protein”, that when in complex with asuitable polynucleotide component, is capable of recognizing, bindingto, and optionally nicking or cleaving all or part of a specificpolynucleotide target sequence.

CRISPR-Cas systems have been classified according to sequence andstructural analysis of components. Multiple CRISPR/Cas systems have beendescribed including Class 1 systems, with multi-subunit effectorcomplexes (comprising type I, type III, and type IV), and Class 2systems, with single protein effectors (comprising type II, type V, andtype VI). A CRISPR-Cas system comprises, at a minimum, a CRISPR RNA(crRNA) molecule and at least one CRISPR-associated (Cas) protein toform crRNA ribonucleoprotein (crRNP) effector complexes. CRISPR-Cas locicomprise an array of identical repeats interspersed with DNA-targetingspacers that encode the crRNA components and an operon-like unit of casgenes encoding the Cas protein components. The resultingribonucleoprotein complex recognizes a polynucleotide in asequence-specific manner. The crRNA serves as a guide RNA for sequencespecific binding of the effector (protein or complex) to double strandDNA sequences, by forming base pairs with the complementary DNA strandwhile displacing the noncomplementary strand to form a so called R-loop.RNA transcripts of CRISPR loci (pre-crRNA) are cleaved specifically inthe repeat sequences by CRISPR associated (Cas) endoribonucleases intype I and type III systems or by RNase III in type II systems. Thenumber of CRISPR-associated genes at a given CRISPR locus can varybetween species.

Different cas genes that encode proteins with different domains arepresent in different CRISPR systems. The cas operon comprises genes thatencode for one or more effector endonucleases, as well as other Casproteins. Some domains may serve more than one purpose, for example Cas9comprises domains for endonuclease functionality as well as for targetcleavage, among others. The Cas endonuclease is guided by a singleCRISPR RNA (crRNA) through direct RNA-DNA base-pairing to recognize aDNA target site that is in close vicinity to a protospacer adjacentmotif (PAM). Class I CRISPR-Cas systems comprise Types I, III, and IV. Acharacteristic feature of Class I systems is the presence of an effectorendonuclease complex instead of a single protein. A Cascade complexcomprises a RNA recognition motif (RRM) and a nucleic acid-bindingdomain that is the core fold of the diverse RAMP (Repeat-AssociatedMysterious Proteins) protein superfamily.

Type I CRISPR-Cas systems comprise a complex of effector proteins,termed Cascade (CRISPR-associated complex for antiviral defense)comprising at a minimum Cas5 and Cas7. The effector complex functionstogether with a single CRISPR RNA (crRNA) and Cas3 to defend againstinvading viral DNA. Type I systems are divided into seven subtypes.

Type III CRISPR-Cas systems, comprising a plurality of cas7 genes,target either ssRNA or ssDNA, and function as either an RNase as well asa target RNA-activated DNA nuclease. Type IV systems, althoughcomprising typical type I cas5 and cas7 domains in addition to acas8-like domain, may lack the CRISPR array that is characteristic ofmost other CRISPR-Cas systems.

Class II CRISPR-Cas systems comprise Types II, V, and VI. Acharacteristic feature of Class II systems is the presence of a singleCas effector protein instead of an effector complex. Types II and V Casproteins comprise an RuvC endonuclease domain that adopts the RNase Hfold. Type II CRISPR/Cas systems employ a crRNA and tracrRNA(trans-activating CRISPR RNA) to guide the Cas endonuclease to its DNAtarget. The crRNA comprises a spacer region complementary to one strandof the double strand DNA target and a region that base pairs with thetracrRNA (trans-activating CRISPR RNA) forming a RNA duplex that directsthe Cas endonuclease to cleave the DNA target, leaving a blunt end.Spacers are acquired through a not fully understood process involvingCas1 and Cas2 proteins. Type II CRISPR/Cas loci typically comprise cas1and cas2 genes in addition to the cas9 gene. Type II CRISR-Cas loci canencode a tracrRNA, which is partially complementary to the repeatswithin the respective CRISPR array, and can comprise other proteins suchas Csn1 and Csn2. The presence of cas9 in the vicinity of cas1 and cas2genes is the hallmark of type II loci. Type V CRISPR/Cas systemscomprise a single Cas endonuclease, including Cpf1 (Cas12) that is anactive RNA-guided endonuclease that does not necessarily require theadditional trans-activating CRISPR (tracr) RNA for target cleavage,unlike Cas9. Type VI CRISPR-Cas systems comprise a cas13 gene thatencodes a nuclease with two HEPN (Higher Eukaryotes and ProkaryotesNucleotide-binding) domains but no HNH or RuvC domains, and are notdependent upon tracrRNA activity. The majority of HEPN domains compriseconserved motifs that constitute a metal-independent endoRNase activesite. Because of this feature, it is thought that type VI systems act onRNA targets instead of the DNA targets that are common to otherCRISPR-Cas systems. To comply with written description and enablementrequirements, incorporated herein by the following references are thefollowing patent publications: 2014/0349405 to Sontheimer; 2014/0377278to Elinav; 2014/0068797 to Doudna; 20200190494 to Hou, et. al.; and2020/0199555 to Zhang; U.S. Pat. No. 9,585,920 to Kovarik; US Pat.Publication No. 20170106026 to Kovarik, and U.S. Pat. No. 9,457,077 toKovarik; US Pat. Publication No. 20170021011 to Kovarik; US Pat.Publication No. 20170173085 to Kovarik.

In certain embodiments, it may be advantageous to genetically modify agut mucosal-associated bacteria with polynucleotides and as taughtherein to express or overexpress the polynucleotides as taught herein orto produce or overproduce the polypeptides, such as butyrate andacetate, directly into the vicinity of, or within the gut mucosalbarrier of a human. In a preferred embodiment, the gutmucosal-associated bacteria may by any bacteria from the species F.prausinitzii, Prevotella intermedia, and/or Akkermansia muciniphilla.Such overproduction may be realized by genetic modification toolsinvolving recombinant DNA technologies, genome editing such as by usingtools based on CRISPR/cas-like systems, or by classical mutationselection systems.

In an embodiment, the genetically modified host cell may be anybacteria, particularly one which is not from a species of bacteria thatnaturally occurs or lives in the vicinity of or within the gut mucosalbarrier of a mammal. Non-limiting examples of such bacteria include anybeneficial isolated intestinal bacterial strains, e.g. probioticbacteria, particularly strains selected from the genera Lactococcus,LactoBacillus, or Bifidobacterium may be used. In addition, strictanaerobic intestinal bacteria may be used such as those belonging to thegenera known to occur in the human intestinal tract. As describedherein, in various embodiments, strictly anaerobic bacteria areencapsulated or microencapsulated to avoid contact with oxygen, and aredelivered to a human such that the encapsulation is dissolved orfractured to release such bacteria in a portion of the body, e.g. gut,where it can thrive.

Certain embodiments employ the bacterium Flavobacterium akiainvivens,which was discovered in 2012 on the plant Wikstroemia oahuensis, or“akia,” which is a flowering shrub endemic to Hawaii. That bacterium hasbeen found on that plant and no other. The bacterium forms 2- to3-millimeter diameter colonies that range from cream to off-white incolor and wet to mucoid in viscosity, and (it) was isolated fromdecaying Wikstroemia oahuensis collected on the island of Oahu.

Certain embodiments are directed to the targeted manipulation of the gutmicrobiome for therapeutic applications, such as the manipulation of thegut microbiome achieved by altering the microbiota population andcomposition, or by modifying the functional metabolic activity of themicrobiome to promote health and restore the microbiome balance. Therehas been recent progress in the engineering of gut commensals, whichalso presents great potential for bio-medical applications.Specifically, in Bacteroides thetaiotaomicron, components for tunablegene expression were developed and characterized and expected functionaloutputs were observed in mice after administration of these engineeredB. thetaiotaomicron. Thus, one aspect of various embodiments is toharness such engineered commensals, especially F. prausntizii for theoverproduction of butyrate, for therapeutic purposes.

F. prausntizii was first isolated in 1922 by C. Prausnitz.Morphologically, F. prausntizii is a Gram-negative, non-motile andnon-sporeforming rod with a diameter of 0.5 to 0.9.times.2.4 to14.0.mu.m. F. prausntizii is a strictly anaerobic bacterium thatproduces butyrate, formate, D-lactate and CO2 but no hydrogen asfermentation products and F. prausntizii growth is inhibited by acidicpH and bile salts. The amount of F. prausntizii in the healthy human gutis linked to diet. Inulin-derived prebiotics have been shown tosignificantly increase F. prausntizii concentration in the gut. F.prausntizii is statistically linked to eight urinary metabolites:dimethylamine, taurine, lactate, glycine, 2-hydroxyisobutyrate,glycolate, 3,5-hydroxylbenzoate and 3-aminoisobutyrate. It is believedthat F. prausntizii has pronounced anti-inflammatory effects. While notbound by theory, F. prausntizii may induce an increased secretion of ananti-inflammatory cytokine interleukin 10, and a decreased secretion ofpro-inflammatory cytokines like interleukin 12 and tumor necrosisfactor—a production. It is further believed that F. prausntizii has theability to suppress inflammation, and it is hypothesized that this isdue to metabolite(s) secreted by F. prausntizii, including but notlimited to butyrate. The number of F. prausntizii is significantlyhigher in the gut of healthy subjects as compared to IBD and it isbelieved that F. prausntizii is crucial to gut homeostasis and diseaseprotection.

F. prausntizii is one of the most abundant bacteria in a healthy humangut and is believed to have a positive effect on the human gut health.F. prausntizii belongs to the Clostridium leptum group (Clostridiumcluster IV), belonging to phylum Firmicutes (Lineage: Bacteria;Firmicutes; Clostridia; Clostridiales; Ruminococcaceae;Faecalibacterium; Faecalibacterium prausnitzii). F. prausntizii has beenpreviously called Fusobacterium prausnitzii (also cited as F.prausntizii), with it only distantly being related to Fusobacteria andmore closely related to members of Clostridium cluster IV.

Moderate butyrate levels can prevent high-fat-diet-induced insulininsensitivity through epigenetic regulation, and mitochondrialbeta-oxidation. F. prausntizii is one of the unique organisms thatreduce various autoimmune diseases, especially type-1 diabetes via themodulation of gut epithelium homeostasis and immune system. Studiesassociated with gut microbiota and type-1 diabetes have a lowerproportion of butyrate-producing organisms, such as Firmicutes andClostridium, which protects against autoimmune diabetes. While not boundby theory, F. prausntizii is believed to regulate the development ofautoimmune diabetes via butyrate dependent complementary pathways. Anabundant quantity of butyrate can lower the gut barrier function andenhance cell apoptosis, with high levels of butyrate stimulating GLP-1secretion and enhancing insulin sensitivity through cAMP signals, suchas PKA and Epac, which inhibit gastric emptying. Due to the inhibitionof gastric emptying, butyrate can be excreted slowly and accumulates,influencing the anti-inflammatory potential, pH, and oxidative stress.

Butyrate is the major product of carbohydrate fermentation in the colon.Butyrate modulates several processes and is a known anti-proliferativeagent. In cultured cell lines, butyrate inhibits DNA synthesis and cellgrowth, mainly by inhibiting histone deacetylase. Butyrate is alsosuggested to regulate the citric acid cycle, fatty acid oxidation,electron transport and TNF-.alpha. signaling. Animal studies haveindicated that butyric acid may have antineoplastic properties, whichmeans that it may protect against colon cancer. As dietary fiber isprotective against colon cancer because carbohydrates entering the largebowel stimulate the production of butyrate. Butyrate has also beensuggested to provide protection against ulcerative. F. prausntizii is animportant producer of butyrate, and the decrease of F. prausntizii hasbeen correlated to lower concentrations of fecal butyrate in healthyhuman subjects and it is believed that F. prausntizii plays an importantrole in the protection of the colon. While not bound by theory, thebenefits of butyrate are thought to depend on several aspects, such astime of exposure and butyrate amount. Increased butyrate production byF. prausntizii is therefore a desired outcome and employment of CRISPRsystems to achieve the same, employing the known genes involved inbutyrate by F. prausntizii is one important embodiment of the presentinvention.

Studies have shown that there was a statistically significant reductionin the F. prausntizii abundance during both fiber-free andfiber-supplemented diets, but it is postulated that the reduction duringthe fiber-supplemented diet was due to the use of pea fiber, which isnot believed to support the growth of F. prausntizii, and thus, with theproper fiber being employed, the increase in butyrate production isachieved. In situations where there is insufficient fiber for thebeneficial bacteria to consume, the bacteria end up eroding the mucus ofthe gut and leads to epithelial access by mucosal pathogens.

The relative abundance of Bacteroidetes and Firmicutes has been linkedto obesity, with the Firmicutes ratio being significantly higher inobese individuals. It is believed that a high number of F. prausntiziileads to higher energy intake, because F. prausntizii is responsible fora significant proportion of fermentation of unabsorbed carbohydrates inthe gut.

F. prausntizii cultivation has proven difficult because the bacterium isa strictly obligatory anaerobe that does not tolerate any oxygen. Asdescribed herein, encapsulation of F. prausntizii is achieved such thatit can be effectively delivered such that the encapsulated structure candegrade or be fractured at an appropriate time and place to release suchbacteria to a human to derive beneficial results, e.g. the increasedproduction of butyrate. For example, microencapsulation, in a xanthanand gellan gum matrix, and a subsequent freeze-drying protocol can beemployed to achieve this result.

In other embodiments, the bacterial composition employed includes bothF. prausntizii and Akkermansia muciniphila, another abundant member ofthe human gut microbiota. It is further believed that Faecalibacteriumprausnitzii plays a vital role in diabetes and can be used as anintervention strategy to treat dysbiosis of the gut's microbialcommunity that is linked to the inflammation, which precedes autoimmunedisease and diabetes.

The microbiota in adults is relatively stable until the persons get 60years old. Gut alterations lead to elevated gut permeability and reducedgut mucosal immunity, contributing to the development of variouscancers, autoimmune disorders, inflammatory bowel diseases, metabolicsyndrome and neurodegenerative diseases. The resultant elevatedintestinal permeability is a consequence of reduced expression of tightjunction proteins that favors the uncontrolled passage of antigens andenables the translocation of bacterial lipopolysaccharide to the gutconnective tissues and to the blood circulation, causing insulinresistance and metabolic endotoxemia.

The gastrointestinal tract pH normally ranges between 5 and 5.5 in theileum and the colon has a range from 6.6 to 7.0, which is one of themain factors in constructing the shape of the microbial communities inthe colon. Diet compositions containing fermentable polysaccharides areregulators of the intestinal pH, which facilitates a more acidicenvironment through the end-products of SCFAs in the gut.

Stool pH becomes more alkaline with the increase in age and differssignificantly between genders with higher consumption of animal proteinbeing one possible mechanism for higher pH. Such alkalinity is generallycaused due to its alkaline metabolites produced by proteolyticputrefactive bacteria, such as Bacteroides, Propionibacterium,Streptococcus, Clostridium, Bacillus, and Staphylococcus.

An individual generally represents a unique collection of genera andsub-species and it may be different based on the diet (vegetarian orWestern with high protein or fat), the age of the host organism, geneticand environmental factors. Diet greatly influences the diversity of themicrobiota in the gut and the microbiota is genetically well equipped toutilize various nutritional substrates to maintain a normal gutmicrobiota pattern. An adequate SOFA (butyrate) production level isessential for gut integrity and butyrate-producing bacteria, such asEubacterium, Fusobacterium, Anaerostipes, Roseburia, Subdoligranulum,and Faecalibacterium, but especially, F. prausntizii, have the potentialof anti-inflammatory effect and help to reduce bacterial translocation,improve the organization of tight junctions and stimulate the secretionof mucin to maintain the integrity of the gut, with beneficial effectsagainst inflammation in the gut.

Inflammation is one of the major pathophysiological factors leading toinsulin resistance and progressively causes type-2 diabetes. F.prausntizii counts significantly decreased in diabetic individuals withnegative correlation to glycated hemoglobin HbA1c values. Along withAkkermansia muciniphila, F. prausntizii is abundantly found inindividuals with normal glucose tolerance compared to the pre-diabeticsubjects. F. prausntizii can convert acetate into butyrate usingbutyryl-CoA: Acetate CoA-transferase (BUT) pathways, thereby providing abalanced pH in the gut.

With the guidance provided herein, as well as the numerous referencesincorporated by reference herein, one of skill in the art willunderstand the feasibility of using engineered bacteria to directlymanipulate the functional output of the microbiota without majormodulation of the microbiota population and composition. Components inthe normal diet and/or employing prebiotics and engineered probioticsare therefore harnessed to render a targeted effect on the host throughmodulating the functional output of the microbiome.

F. prausntizii is a multi-skilled commensal organism and a chief memberof human microbiota. It is broadly distributed in the digestive tract ofmammals and also in some insects. It is rich in the hind gut rather thanin the stomach, as well as jejunum. The consumption of a higher quantityof animal meat, animal fat, sugar, processed foods, and low fiber diet(the typical westernized diet) reduces the count of F. prausntizii,while a high-fiber (vegetables and fruits) and low meat diet enhance thecount of F. prausntizii. It is known to consume a variety of dietcontaining polysaccharides, such as the prebiotic inulin, arabinoxylans,apple pectin, oligofructose, resistant starch, fructan supplement,pectins and some host-derived carbon sources (including d-glucosamineand N-Acetyl-d-glucosamine). Meta-analyses also show that the increasedconsumption of fiber significantly reduces the risk of mortality.

The discovery of the clustered regularly interspaced short palindromicrepeats (CRISPR) and the CRISPR-associated nuclease 9 (Cas9) system, hasled to an array of strategies to manipulate the gut microbiome withprecision. Engineered phage (with the CRISPR-Cas9 system) can beemployed to target pathogenic bacteria, or remove a population ofbacteria that aids pathogenic bacterial growth, thereby fine-tuning andrestoring the balance of the gut microbiome. CRISPR/Cas9 can also beused to manipulate and differentiate genetically heterogeneous bacteria,even of the same species. Unlike conventional drugs, the CRISPR/Cas9system targets specific bacteria at the gene level to selectively removepathogens, virulence factors, genes of undesired expressed proteins,etc. and can further be used as an antimicrobial adjuvant to improveantibiotic treatment. Citorik et. al. demonstrated how CRISPR/Cas9 canbe delivered using bacteriophages, targeting the ndm-1 gene, which codesfor the broad-spectrum carbapenemase, New-Delhimetallo-.beta.-lactamase. Ndm-1 targeting CRISPR/Cas9 specificallyeliminated E. coli harboring the gene without affecting wild-type, orother, E. coli strains present in a synthetic consortium of microbes.Other examples include the re-sensitization of bacteria to antibioticsand immunization of bacteria to incoming plasmids conferring antibioticresistance using temperate phages. Yosef et al. used CRISPR/Cas9 totarget ndm-1 and ctx-M-15, which expresses a broad-spectrumbeta-lactamase, and effectively selected the transduced bacteria thatwere antibiotic-sensitive. Thus, CRISPR/Cas9 may be employed tomanipulate the gut microbiome by discriminating at the gene level tochange the characteristics and functional output of the gut microbiomefor therapeutic applications.

In particular embodiments of the present invention, the bacterialformulation may include bacteria selected from the group consisting ofNitrosomonas eutropha and Propionibacterium. More particularly, theequilibrium of a bacterial population of the region of the skin of theindividual is modified to increase the number of Propionibacteriumbacteria and to decrease the number of Staphylococcus bacteria on theindividual's skin in such region. In other embodiments, the bacterialformulation includes the bacteria Staphylococcus aureus that has beenmodified by employing a CRISPR-Cas or Cpf1 system to interfere with S.aureus virulence regulation involving the Agr quorum-sensing signalingmolecule. In several embodiments, the bacterial formulation comprises abacteria that has a tropism specific for the human species. In others,the bacterial formulation comprises at least two of the bacteriaselected from the group consisting of: Prevotella; LactoBacillusjohnsonii; Bacteroides fragilis, LactoBacillus ruminus and L.infantitis. In certain embodiments the bacteria is an ammonia oxidizingbacteria. In other embodiments, the region of the skin to which thebacterial formulation is applied is the scalp. In various embodiments,rather than using a wild-type bacteria, the bacteria employed is onethat has been modified by CRISPR-Cas or CRISPR-Cpf1 to delete afunctional virulence factor from the bacteria. In particularembodiments, the method includes administering to the skin a bacteriathat produces tomatidine. In others, the bacteria produces p53. Thus, insome embodiments, the method involves use of bacteria wherein aCRISPR-Cas or CRISPR-Cpf1 system is employed to insert a gene for theproduction of tomatidine and/or p53 into at least one of the bacteria inthe bacterial formulation. In others, a CRISPR-Cas or CRISPR-Cpf1 systemis employed to insert one or more genes into the bacteria comprising thebacterial formulation to facilitate the oxidizing of ammonia by thebacteria. To further enhance the ability of desired bacteria to bemaintained on the skin of an individual, certain methods furthercomprise administering to the individual's skin a prebiotic thatcomprises a nutrient source for the bacteria that is assimilated by thebacteria, and preferably one that is not digestible by the individual.In particular embodiments, the method further includes administering tothe skin an extract derived from a helminth selected from the groupconsisting of Capillaria hepatica, Dicrocoelium dendriticum, Ascarislumbricoides, Enterobius vermicularis, Trichuris trichiura, Ancylostomaduodenale, Necator americanus, Strongyloides stercoralis, Haemonchuscontortus, and Trichinella spiralis. In still others, the bacterialformulation includes at least one arabinogalactan. Yet others include atleast one of the following: L. infantitis, and L. johnsonii. In aparticular embodiment, the bacterial formulation includes at least onebacteria modified via a CRISPR-Cas system to express a gene encodinginterferon regulatory factor 4.

As for lotions of the present invention, in preferred embodiments, thereis an objective to limit if not preclude the use of phthalates, whichare extremely toxic and are believed to also be human carcinogens. Thus,in preferred embodiments of the present invention, such lotions do notemploy such toxic agents, and in particular agents toxic to bacterialspecies for which the inventors suggest be used, e.g. those modified toreduce pathogenicity, virulence factors, etc, so as to establish apopulation of such modified bacteria on a person's skin, and in such amanner, reduce the incidence of skin infections and diseases. Thus,lotions, creams, gels, etc. that include such toxic agents, includingbut not limited to phthalates, are not employed, but rather, lotionsthat provide an environment for the bacteria as set forth herein tosurvive and to thus be available to provide benefits to the skin ofindividuals to which they are applied, are particularly preferred.

Healthy, normal skin exhibits a slightly acidic pH in the range of4.2-5.6, which aids in the prevention of pathogenic bacterialcolonization, regulation of enzyme activity, and maintenance of amoisture-rich environment; however, after the age of 70, the pH of skinrises significantly, stimulating protease activity. Thus, one objectiveof several embodiments of the present invention is directed to loweringthe pH of the skin of an individual, especially those at about the ageof 70, so as to encourage a skin environment conducive to theproliferation of one or more bacteria that have been modified to promoteskin health and to reduce the ability of undesired bacteria fromcolonizing the skin of the person. Probiotic metabolism frequentlyproduces acidic molecules, lowering the pH of the surroundingenvironments seen with Lactobacilli producing free fatty acids (FFAs)and conjugated linoleic acid (CLA) during the fermentation process.Thus, the use of probiotics is employed to restore the normal skin pHand consequently return protease activity levels closer to those seen inyoung, healthy skin.

The main microbes that reside on human skin can be divided into fourphyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes.Staphylococcus spp. and Corynebacterium spp. are the dominant bacteriaat the genus level. Significantly fewer Corynebacterium spp. have beenobserved in cachexia patients compared to healthy subjects. Theseresults suggest that the presence of cancer and cachexia alters humanskin bacterial communities. Understanding the changes in microbiotaduring cancer cachexia may lead to new insights into the syndrome.

Competitive inhibition is relied upon in various embodiments to advancethe repopulation of skin with beneficial microbes. In one embodiment,repopulating an individual's skin with beneficial bacteria, preferablyin balanced percentages and having preferred species provided, can beused in conjunction with an antimicrobial composition. Preferably, anantimicrobial is first administered to suppress or eradicate theresident populations of bacteria on a person's skin, including anyabnormal organisms or pathogenic bacteria, then the normal flora isrepopulated by the administration of at least one of the modifiedbacteria as described herein, including those modified using CRISPR-Casand/or Cpf1 systems to delete certain portions of genes or to addcertain genes to facilitate the colonization of a person's skin withbeneficial bacteria that maintain the general health of a person's skin.

In various embodiments, cosmetics are provided that provide for a mediumfavorable for maintaining a desired physico-chemical balance of the skinwithout favoring the development of pathogenic microorganisms. Toachieve this objective, certain oligosaccharides that are metabolized byseveral beneficial strains of the skin microflora, such as Micrococcuskristinae, Micrococcus sedentarius, Staphylococcus capitis,Corynebacterium xerosis and LactoBacillus pentosus, are employed informulations, in conjunction with one or more of the modified bacteriaas described herein. In particular embodiments, oligosaccharides areemployed in formulations for the skin that include one or more ofLactoBacillus pentosus, Micrococcus kristinae, Gardnerella vaginalis,Propionibacterium avidum and Propionibacterium granulosum. As statedherein above, it is often beneficial to further acidify the culturemedium, and this can be achieved, for example, by employing Lactobacillito produce in particular lactic acid to achieve pH reducing effects.

In certain embodiments, the present invention is directed to cosmeticcompositions having at least one oligosaccharide chosen from the groupconsisting of gluco-oligosaccharides, fructo-oligosaccharides, andgalacto-oligosaccharides and mixtures thereof. In addition to theoligosaccharide constituent, the cosmetic compositions of particularembodiments of the invention may contain other ingredients, but cautionis warranted as one objective is to avoid incorporating ingredientswhose properties would interfere with the development of the beneficialskin microflora and the preservation of acidic conditions. Thus, it isadvisable to avoid incorporating bactericidal ingredients in proportionswhich would annihilate the endogenous microflora, or ingredients whichconfer a pronounced basic character on the composition. For example, inpreferred embodiments, reduction if not elimination of ionicsurface-active agents, such as sodium lauryl sulfate, is advisable, aswell as other well known agents having bactericidal properties. Instead,use of a non-ionic surface-active agent such as an alkyl glucoside or adialkyl ester may be employed in various embodiments. Preferably,cosmetic compositions of the invention contain an acidic buffer whichadjusts the pH of the composition to about pH 4 to 7 range, preferablyabout 5 to 6.5 pH. At such range, especially on the lower side,mutualistic flora such as Staphylococci, Micrococci, Corynebacterium andPropionibacteria preferably grow but not transient bacteria such as Gramnegative bacteria like Escherichia and Pseudomonas or Gram positive onessuch as Staphylococcus aureus or Candida albicans.

Certain other embodiments are directed to the rebalancing of the skinmicrobiota using antimicrobials with selective action. For example, incertain embodiments a balance of species and characteristics is soughtto provide skin formulations that maintain a well-balanced bacterialflora, and especially one that includes one or more of the modifiedbacteria as described herein. Thus, one particular aspect of variousembodiments is directed to the provision of embodiments targeted toreduce undesired body odor (and in various embodiments, activelyprovides microbes that generate desired odors and reduces the effects ofmalodors by other bacteria) which can be gender specific.

In various formulations of the present invention, the use of bacteriaable to generate lactic acid to serve as a moisturizing factor, stillothers that produce hyaluronic acid to improve skin hydration andelasticity, and that include sphingomyelinase to generate ceramide toenhance skin barrier function, are preferred compositions. One aspect ofthe present invention is directed to restoring homeostasis to treatcertain skin diseases by remedying the dysbiosis in the skin habitat byestablishing a desired colony of various diverse bacteria, especiallythose modified as described herein to establish and maintain a healthyskin condition on an individual's skin.

In one embodiment of the present invention, bacteria species areemployed that have been modified via CRISPR-Cas systems to reducedmalodor without the employment of aluminum or zirconium salts. Suchmodified bacteria suppress malodor and counteract or suppress sweatmalodor. Even more preferred bacteria have been modified to expresscompounds of a pleasant and desirable scent. Such bacteria can thusprovide amounts of a perfume scent that is pleasant to a person and thatcan at least partially mask the unpleasant body odor smells produced bya person. Splicing in such “perfume” genes into bacteria using theCRISPR-Cas system is one way to accomplish this objective. Use of suchbacteria on a person's skin, and in particular under armpits where theparticular type of bacteria is selected to grow and out-complete othermicrobes in such a moister environment (as compared to elbows, etc.) canbe used to enhance the desired smells of one's body while limiting theamount of traditional antiperspirants and deodorants conventionallyemployed. Still other embodiments include the use of bacteria thatutilize as their food source the very bacteria that produce malodors. Insuch a fashion the desired bacteria feed off of the products produced byundesired bacteria on a person's skin, and in particular under anindividual's arm, so that undesired body odor is reduced and without theuse of traditional chemicals and compounds as previously discussed.

To further comply with written description and enablement requirements,the following patents and patent publications are also incorporatedherein by this reference in their entireties: are the following: U.S.Pat. No. 8,815,538 to Lanzalaco, et al.; 20150374607 to Lanzalaco, etal.; 20150361436 to Hitchcock et al.; 20150353901 to Liu et al.; U.S.Pat. No. 5,518,733 to Lamothe, et al.; 20150259728 to Cutliffe et al.U.S. Pat. No. 8,685,389 to Baur; 20140065209 to Putaala et al.; U.S.Pat. No. 8,481,299 to Gueniche; WO 2011029701 to Banowski; 20150071957to Kelly; 20150202136 to Lanzalaco; 20150017227 to Kim; U.S. Pat. No.7,820,420 to Whitlock; 20150202136 to Lanzalaco et al.; U.S. Pat. No.5,518,733 to Lamothe, et al.; U.S. Pat. No. 8,815,538 to Lanzalaco et.al; U.S. Pat. No. 8,951,775 to Castiel; WO 2006/07922; U.S. Pat. No.9,234,204 to Qvit-Raz et al.; U.S. Pat. No. 8,758,764 to Masignani, etal.; U.S. Pat. No. 9,028,841 to Henn et al.; 20160008412 to Putaala etal., 20150064138 to Lu; 20150017227 to Kim; United States PatentApplication No. 20160314281 to Apte; 20160151427 to Whitlock et al.;20140044677 to Raz et al.; 20160168594 to Zhang et al. U.S. Pat. Nos.7,267,975; 9,288,981; United States Patent Application No. 20160122806;U.S. Pat. No. 9,234,204 to Noga Qvit-Raz; US20120301452; 20160271189 toCutcliffe; US Pat. Applic. No. 2008242543; 20160040216 to Wilder; andUnited States Patent Application No. 20160089315 to Kleinberg, et al.,20070148136 to Whitlock et al., 20190059314 to Aharoni; 20200009268 toScholz and 20200009185 to Shin;

In certain embodiments, one aspect of the present invention is directedto the treatment of acne by using probiotic treatments that includeeffective amounts of Staphylococcus epidermidis and/or LactoBacillusplantarum to inhibit P. acnes growth, which are believed to producesuccinic acid, shown to inhibit P. acnes growth. CRISPR-Cas and/or Cpf1systems are used to modify such bacteria in a manner that reduces theoccurrence of acne, such as by altering the expression of genes so thatthe amount of succinic acid on a person's skin is increased.

Certain aspects of the present invention relate to a compositionincluding ammonia oxidizing bacteria to increase production of nitricoxide and/or nitric oxide precursors in close proximity to a person'sskin. More specifically, applying a composition of an ammonia oxidizingbacteria to skin during or after bathing to metabolize urea and othercomponents of perspiration into nitrite and ultimately into Nitric Oxide(NO) results in a natural source of NO. One aspect of the presentinvention causes topical nitric oxide release at or near the surface ofthe skin where it can diffuse into the skin and have local as well assystemic effects. This naturally produced nitric oxide can thenparticipate in the normal metabolic pathways by which nitric oxide isutilized by the body. Adding urea or ammonium salts to the skin providesadditional substrates that these bacteria utilize to form nitrite. Asused herein, the phrase near the surface is defined as adjacent to or inclose proximity to, but need not be in contact with the surface.

In still other embodiments, CRISPR systems are used to modify the generaPropionibacterium, Corynebacterium and Staphylococcus, and in particularS. epidermidis, which are among the most common groups on a person'sskin, with such modifications making such species more amenable togrowth on the skin, thus providing for competitive inhibition ofnon-modified bacteria on the skin. As one of skill in the art willappreciate, a suitable topical composition comprising a population ofthe above bacteria can be, in various embodiments, a cream, lotion,emulsion, gel, ointment, liquid or spray. In one embodiment, the topicalcomposition is formulated to provide at least about 10.sup.2 bacteriaper cm.sup.2. In another aspect, a method of treatment is provided,wherein a composition as described herein is topically applied to theskin and in certain embodiments, topically applying includes topicallyapplying to a mucosal surface (nasal, vaginal, rectal, oral surfaces) ofa person. A suitable lotion may also include amounts of sugars that thevarious LactoBacillus microorganisms may assimilate to survive andthrive. These sugars and life bacteria-supporting compounds are known tothose in the art and as otherwise referenced in various incorporatedwritings. In still other embodiments, pulverized compositions ofhelminth collections and bacteria preferably obtained from Amish-soils,may be employed in various administrative modes, including but notlimited to lotions, creams, and other topical applications.

Because skin cells turn over every 4 weeks, differentiating from stemcells deep within the epidermis and hair follicles, they eventuallyslough off from the upper layer as cornified (enucleated, dead) cells.The skin microbiome is vastly different from the gut microbiome, whichconsists primarily of members of Firmicutes and Bacteroidetes divisions.The skin is also different from the gut in that there is a low level ofinterpersonal variation of skin microbiomes, which is not the case ingut studies. Regardless, there is a low level of deep evolutionarylineage diversity, with only six of the more than 70 described bacterialdivisions associated with the skin, and approximately the same numberfor the gut, which compares to a vast array of bacteria in soil.

A subject of the invention is also the topical use of an effectiveamount of at least one probiotic microorganism according to theinvention, especially of the LactoBacillus and/or Bifidobacterium sp.Genus, and in particular of the LactoBacillus paracasei ST11 strain, toreduce the likelihood of seborrhoeic dermatosis associated with oilyskin or skin with an oily tendency. Microorganisms suitable for thisaspect of the invention include an ascomycetes, such as Saccharomyces,Yarrowia, Kluyveromyces, Torulaspora, Schizosaccharomyces pombe,Debaromyces, Candida, Pichia, Aspergillus and Penicillium, bacteria ofthe genus Bifidobacterium, Bacteroides, Fusobacterium, Melissococcus,Propionibacterium, Enterococcus, Lactococcus, Staphylococcus,Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc,Weissella, Aerococcus, Oenococcus and LactoBacillus, and mixturesthereof.

As ascomycetes is particularly suitable for particular embodiments ofthe present invention, one may desire the use of Yarrowia lipolitica andKluyveromyces lactis, as well as Saccharomyces cereviseae, Torulaspora,Schizosaccharomyces pombe, Candida and Pichia, all of the samepreferably modified via CRISPR-Cas or Cpf1 systems to reduce virulencefactors associated with the same. Specific examples of probioticmicroorganisms also suitable for the invention include: Bifidobacteriumadolescentis, Bifidobacterium animalis, Bifidobacterium bifidum,Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum,Bifidobacterium infantis, Bifidobacterium pseudocatenulatum,LactoBacillus acidophilus (NCFB 1748); LactoBacillus amylovorus,LactoBacillus casei (Shirota), LactoBacillus rhamnosus (strain GG),LactoBacillus brevis, LactoBacillus crispatus, LactoBacillusdelbrueckii(subsp bulgaricus, lactis), LactoBacillus fermentum,LactoBacillus helveticus, LactoBacillus gallinarum, LactoBacillusgasseri, LactoBacillus johnsonii (CNCM 1-1225), LactoBacillus plantarum,LactoBacillus reuteri, LactoBacillus salivarius, LactoBacillusalimentarius, LactoBacillus curvatus, LactoBacillus casei subsp. casei,LactoBacillus sake, Lactococcus lactis, Enterococcus (faecalis,faecium), Lactococcus lactis (subsp lactis or cremoris), Leuconostocmesenteroides subsp dextranicum, Pediococcus acidilactici,SporoLactoBacillus inulinus, Streptococcus salvarius subsp.thermophilus, Streptococcus thermophilus, Staphylococccus carnosus,Staphylococcus xylosus, Saccharomyces (cerevisiae or else boulardii),Bacillus (cereus var toyo or subtilis), Bacillus coagulans, Bacilluslicheniformis, Escherichia coli strain nissle, Propionibacteriumfreudenreichii, and mixtures thereof. In other embodiments, probioticmicroorganisms for use in the invention are derived from the group oflactic acid bacteria, such as, in particular, LactoBacillus and/orBifidobacterium. In particular, various embodiments use lactic acidbacteria such as LactoBacillus johnsonii, LactoBacillus reuteri,LactoBacillus rhamnosus, LactoBacillus paracasei, LactoBacillus casei orBifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum,Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacteriuminfantis, Bifidobacterium adolescentis, Bifidobacteriumpseudocatenulatum, and mixtures thereof. Most preferably for particularembodiments, CRISPR modified bacteria of the following are employed:LactoBacillus johnsonii, LactoBacillus paracasei, Bifidobacteriumadolescentis and Bifidobacterium longum, respectively depositedaccording to the Treaty of Budapest with the Institut Pasteur (28 rue duDocteur Roux, F-75024 Paris cedex 15) on 30 Jun. 1992, 12 Jan. 1999, 15Apr. 1999 and 15 Apr. 1999 under the following designations: CNCM1-1225, CNCM I-2116, CNCM 1-2168 and CNCM 1-2170, and theBifidobacterium lactis (Bb 12) (ATCC27536) or Bifidobacterium longum(BB536) genus. The Bifidobacterium lactis (ATCC27536) strain can beobtained from Hansen (Chr. Hansen A/S, 10-12 Boege Alle, P.O. Box 407,DK-2970 Hoersholm, Denmark); LactoBacillus paracasei ST11 straindeposited according to the Treaty of Budapest with the Institut Pasteur(28 rue du Docteur Roux, F-75024 Paris cedex 15) on 12 Jan. 1999 underthe designation CNCM 1-2116, and/or a fraction thereof and/or ametabolite thereof.

According to one variant embodiment, the invention relates to the use,in addition to a first probiotic microorganism, as defined above,especially of the LactoBacillus and/or Bifidobacterium sp. genus, of atleast an effective amount of at least a second microorganism, distinctfrom said first microorganism. Such a second microorganism may be anascomycetes, such as Saccharomyces, Yarrowia, Kluyveromyces,Torulaspora, Schizosaccharomyces pombe, Debaromyces, Candida, Pichia,Aspergillus and Penicillium, bacteria of the Bacteroides, Fusobacterium,Melissococcus, Propionibacterium, Enterococcus, Lactococcus,Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus,Leuconostoc, Weissella, Aerococcus, Oenococcus, LactoBacillus orBifidobacterium genus, and mixtures thereof.

In other embodiments, CRISPR-Cas and or Cpf1 systems are used to modifyat least one of Enterobacter aerogenes, Acinetobacter baumannii, andKlebsiella pneumoniae, which are three gram negative bacteria commonlyfound on the skin, and which utilize fatty acids in a manner thataffects bacterial phenotype. The modifications to such bacteria includethose effective in enhancing the beneficial traits of such bacteria fora person's skin and the reduction of respective virulence factors of thebacteria. In such a manner, one aspect of the present invention is tomaintain a microbiome in a healthy, balanced state and/or returning amicrobiome to a balanced state by providing certain desirablemicroorganisms with sufficient nutrients to thrive, and therebyoutcompete and/or kill the undesirable bacteria. It has been found thatCorynebacterium jeikeium (“C. jeikeium”), Staphylococcus epidermidis(“S. epidermidis”), and Propionibacterium acnes (“P. acnes”), present onboth the face and forearms of humans, can be used to address dry skinconditions and diseases on such tissues. Modifications of virulencefactors of pathogenic bacteria associated with such conditions, as wellas combining such modified bacteria with other commensal microorganisms,is one aspect of the present invention. Such bacteria include: Alphaproteobacteria, Beta proteobacteria, Gamma proteobacteria,Propionibacteria, Corynebacteria, Actinobacteria, Clostridiales,Lactobacillales, Staphylococcus, Bacillus, Micrococcus, Streptococcus,Bacteroidales, Flavobacteriales, Enterococcus, and Pseudomonas.

Various embodiments of the present invention are directed to a methodfor reducing the likelihood of the onset of a disease, such as cancers,by administering to a subject a therapeutically effective amount of acomposition comprising a probiotic microorganism, rather than attemptingto alter the eukaryotic genome of the individual. It is believed that bymerely modifying a person's microbiome, whether it be their gut, oral orskin microbiome, it is possible to treat, if not protect suchindividuals from a vast array of previously devastating diseases of man.For example, Helicobacter species have been associated with enhancedcarcinogenesis including liver cancer, colon cancer, and mammarycarcinoma. Probiotic formulations containing lactic acid bacteria havebeen shown to reduce the incidence of chemically mediated hepatocellularcarcinoma and colon cancer. Bacteria that have been modified using aCRISPR-Cas system to purposefully excise or interfere with virulencefactors of particular pathogenic bacteria, and the employment of suchmodified bacteria to adjust the population of a person's microbiome, isan effective way to treat a vast number of historically difficultdiseases.

The balance between health and disease is imperiled by infections. Whenimmunity is lowered, the human body is less able to eradicate cancercells, which would otherwise be kept in check. In certain embodiments, amushroom component is also employed to achieve desired health effects.For example, in various embodiments, the mushroom mycelium is used toprotect against viruses that cause disease in humans, such as thosemushrooms derived or obtained from Antrodia, Fomes, Fomitopsis,Ganoderma, Inonotus, Schizophyllum, Phellinus, Piptoporus, Trametes andother taxa in the Polyporaceae. Ethyl alcohol/water extractiontechniques are employed on living mycelium to obtain antiviral compoundsand that are effective to reduce viruses that cause inflammation andimmune deactivation which are contributory to oncogenesis. Such extractsreduce the pathogenicity of viruses and by doing so, reduce cancer riskand also significantly enhance the benefits of other anticancer drugs toincrease the quality of life of cancer patients. Used in combinationwith the various other aspects of the present invention, including thebeneficial modified bacterial species as described herein, a person'soverall health is improved by reducing the chances of infection,inflammation and cancer, by improving and adjusting the microbiome ofindividuals and by having certain mushroom derived compoundsadministered, (some of which can be inserted into the genome of bacteriavia the CRISPR-Cas system) such that beneficial compounds areadministered to individuals to prevent and treat various diseases, suchas but not limited to, cancer.

In particular embodiments, a method of the present invention involves amethod of improving the health of a person's skin microbiome byidentifying a skin region to be treated in terms of age, ethnicity,region of the body and age of the person and then applying a skincommensal prebiotic agent adapted to address the skin region; whereinthe prebiotic comprises at least one microbe that has been modified by aCRISPR-Cas or Cpf1 system to add or delete a gene that enhances thehealth of a person's skin.

Other embodiments include a method of improving the health of a person'sskin microbiome, comprising: providing a first type of bacteria to aperson's skin that produces an agent that another second bacterialspecies requires for growth; after applying said first bacteria to theskin of a person, then applying the second bacteria to the person'sskin, wherein both the first and the second bacteria comprise at leastone microbe that has been modified by a CRISPR-Cas or Cpf1 system to addor delete a gene that enhances the health of a person's skin. In stillothers, the virulence factor of the first bacteria is modified viaCRISPR-Cas to impede the interaction of bacterial adhesions andkeratinocyte receptors. One can modify the expression of at least onegene by employing a CRISPR-Cas system to decrease the pathogenesis of askin infection. Moreover, one can employ a second bacteria whose growthon a person's skin is enhanced by at least 2-fold when in the presenceof the first bacteria, wherein the second bacteria is modified viaCRISPR-Cas to have an essential growth required component deleted fromits genome, and wherein the first bacteria has been modified viaCRIPSR-Cas to add the same essential growth component that the secondbacteria requires for growth.

Existing antibiotic therapies non-specifically kill the majority ofskin-residing bacteria, disrupting the homeostasis of skin residentmicroflora. For example, benzoyl peroxide (BPO) is one of the mostfrequently used topical medications. BPO strongly suppresses the growthof S. epidermidis. S. epidermidis contributes to the skin residentmicroflora-based defense of the skin epithelium. The imbalance ofmicroflora is believed by the present inventor to contribute to thepathogenesis of skin inflammatory diseases, such as atopic dermatitis,rosacea and acne vulgaris etc. Thus, in various embodiments, suchantibiotic therapies are not employed but instead, beneficial bacteriaare administered to a person's skin in a manner that beneficial resultsare achieved (e.g. reduction in malodors, generation of desired odors bybacterial production of scents, etc.) CRISPR-Cas systems are preferablyemployed to modify species of bacteria already found on an individual'sskin such that the disturbance of the “normal” population of aparticular person is not disturbed in a fashion that could lead todisease or discomfort.

Various embodiments include providing two or more bacteria species thatare normally found on a person's skin, and modifying the same to removevirulence factors via CRISPR; including in such bacteria beneficialgenes for the production of emollients, lipids, scents, etc. and usingcompetitive inhibition to foster the growth of bacteria purposefullyexposed to the skin surface so that pathogenic bacteria are notpermitted to establish and grow. In certain embodiment, CRISPR isemployed to insert a gene for the production of tomatidine in a bacteriasuch that, especially in the gut microbiome, but preferably also in theoral and skin microbiome, tomatidine is expressed. Tomatidine has theeffect of increasing and enhancing muscle performance and in maintainingthe weight, especially muscle mass, of an individual.

Staphylococcus aureus is the most pathogenic species of theStaphylococcus genus, responsible for food poisoning, suppurativelocalized infections and physical septicemia (graft, cardiacprostheses). Ogston (1881) coined the genus Staphylococcus to describegrapelike clusters of bacteria (staphylogrape, Gr.) recovered in pusfrom surgical abscesses. The species proves to be an opportunisticpathogen in certain locations or under certain circumstances and isfound in the commensal flora (in 15% to 30% of healthy individuals inthe nasal fossae). S. aureus has pathogenic capacities, in particular aninvasive capacity, a capacity to multiply and to spread in the organism,and also a toxic capacity. S. aureus has a great capacity for developingantibiotic-resistant mutants. In one embodiment, modified Staphylococcusepidermidis is used to produce enhanced amounts of anti-microbialpeptides that inhibit S. aureus biofilm formation, with preferredembodiments employing CRISPR-Cas systems to achieve such modifications.

In various embodiments, due to the inclusion of bacteria-hostileformulations in over-the-counter lotions and related products, the useof conventional lotions is not suggested for employment in conjunctionwith the administration of many embodiments of the present invention.Lotions presently available are believed to be counterproductive to thefostering the beneficial growth of beneficial bacteria on a person'sskin. E.g. salicylic acid is bacteriostatic that limits the growth ofbacteria by interfering with bacterial protein production by downregulating fitness and virulence factor production of bacteria. As it isknown that gram positive and gram negative bacteria prefer slightlybasic conditions pH 7.5 and warm temperatures 37 degrees Celsius (98.6degrees Fahrenheit), the establishment and maintenance of slightlyacidic conditions on one's skin is a preferred objective and is achievedby the fostering of certain bacteria that produce lactic acid on aperson's skin.

All gram negative bacteria are disease producing. As such, one aspect ofthe present invention is directed to reducing the number of gramnegative bacteria on a person's skin by adjusting the overall local pHof the skin tissue region by providing bacterial species that areselected to synergistically grow together and establish a desired pHlevel that discourages the growth of gram negative bacteria on the skin.Caution is called for, however, as the pH should not get too low, asfungi, yeast, and molds prefer acid conditions (pH 5.5-6) at roomtemperature to multiply. In this regard, the pH is preferablymaintained, either by bacterial species producing lactic acid at amountssufficient to achieve such levels, or by other pH adjustment methods, inorder to hinder the growth and progression of pyogenic cocci, sphericalbacteria that cause various suppurative (pus-producing) infections.Included are the Gram-positive cocci Staphylococcus aureus,Streptococcus pyogenes and Streptococcus pneumoniae, and theGram-negative cocci, Neisseria gonorrhoeae and N. meningitidis. In termsof their phylogeny, physiology and genetics, these genera of bacteriaare unrelated to one another. They share a common ecology, however, asparasites of humans. The Gram-positive cocci are the leading pathogensof humans. It is estimated that they produce at least a third of all thebacterial infections of humans, including strep throat, pneumonia,otitis media, meningitis, food poisoning, various skin diseases andsevere types of septic shock. The Gram-negative cocci, notably theNeisseriae, cause gonorrhea and Meningococcal meningitis. Again, thereduction of virulence factors of such bacteria via CRISPR-Cas or Cpf1systems reduces the incidence of infections caused by such bacteria andleads to methods and systems for establishing and maintaining a healthyskin microbiome, free of disease.

In yet other embodiments, bacteria are modified to express certaincompounds that deter mosquitoes from alighting on an individual's skin.In certain embodiments bacteria are modified to produce amounts of DEET,with such bacteria being contacted to an individual's skin. In stillother embodiments other known insect repellents such as eucalyptol,linalool, and thujone, are expressed by such bacteria to deter insects.In still other embodiments, bacteria are modified to express a proteinmember of the ionotropic receptor family, IR40a, which is a DEETreceptor. In addition, other repellent proteins structurally related toDEET may be employed to repel insects, such as mosquitoes and flies.

One aspect of various embodiments is directed to the expression ofparticular phytochemicals by CRISPR-Cas modified bacteria to amelioratea human disease. Phytochemicals exert their antibacterial activitythrough different mechanisms of action, such as damage to the bacterialmembrane and suppression of virulence factors, including inhibition ofthe activity of enzymes and toxins, and bacterial biofilm formation.These antibacterial effects of phytochemicals may be due to the presenceof one or more of alkaloids, sulfur-containing phytochemicals,terpenoids, and polyphenols and also may involve a synergistic effectwhen used in combination with conventional antibiotics, thus modifyingantibiotic resistance.

Treatments for various types of cancer are desired that relate to theproduction of competently folded p53 tumor support factor. There hasbeen a long felt but unmet need for a way to inexpensively administerdesired amounts of p53 protein to an individual in need thereof. Thepresent invention in several of its aspects addresses this concern, forexample, by the expression of p53 by human microbiome bacteria. Incertain embodiments of the present invention, a method for treatingcancer cachexia involves the administering to the microbiome of asubject in need thereof an effective amount of a bacterial combinationthat expresses p53 protein and tomatidine, such cancer being forexample, one of breast cancer, bladder cancer, kidney cancer, throat,oral, brain cancer, or colorectal cancer. In certain embodiments, thecancer is a metastatic cancer; and the microbiome is one or more of thegut microbiome, the oral microbiome (including the nasal microbiome) orthe skin microbiome. Other embodiments involve mucosally administeringto the subject an effective amount of a bacteria that has been modifiedto express a particular protein or drug or compound, especially thosethat are anti-cancer agents, such as one of tomatidine and p53, with thebacteria selected from the group consisting of—Streptococcus,Actinomyces, Veillonella, Fusobacterium, Porphromonas, Prevotella,Treponema, Neisseria, Haemophilus, Eubacteria, Lactobacterium,Capnocytophaga, Eikenella, Leptotrichia, Peptostreptococcus,Staphylococcus, Streptococcus thermophilus and Propionibacterium. Stillother embodiments include the provision of Streptomyces hygroscopicus inan amount effective to produce therapeutically effective amounts ofrapamycin to the subject. Providing the genes sufficient to makerapamycin and including them in a suitable microbe, preferably one ofthe bacteria listed herein, is one method for providing rapamycin to anindividual in a manner such that the “bugs as drugs” administration canbe achieved. One of ordinary skill in the art will appreciate how toselect the genes responsible for the generation of rapamycin so as toachieve expression thereof in a fashion that does not kill the microbebeing employed to manufacture therapeutically sufficient and desiredamounts of rapamycin. The genetic sequence of the genes involved in theproduction of rapamycin by Streptomyces hygroscopicus.

Incorporated by reference herein are the following to address writtendescription and enablement issues: US Pat. Publication No. 20190388471to June; 20190000815 to Melin; 20180258100 to Gregory; 20170027914 toQi; 20130310416 to Blagosklonny.

It should be appreciated that a therapeutically effective amount ispreferably an amount sufficient to elicit any of the listed effects ofnatural tomatidine, rapamycin and/or p53, for example, including, butnot limited to, the power to treat cancer cachexia in a fashiondemonstrated by a result indicating the maintenance of muscle mass inthe individual treated. In preferred embodiments, the mucosaladministration is oral administration and the subject individualmaintains or increases muscle mass. In most preferred embodiments, thebacterial composition has been modified via a CRISPR-Cas or CPf1 systemto express a desired protein or compound, such as tomatidine, p53,rapamycin, etc., and in other embodiments, produces both tomatidine andp53 protein. Other embodiments include a bacterial composition thatincludes one of a Chlamydia species, or Shigella flexneri, Mycoplasmabacteria, and H. pylori.

In the 1920s, Dr Otto Warburg first suggested the significant differencein energy metabolism between malignant cancer cells and adjacent normalcells. Tumor cells mainly adopt the glycolysis as energy source tomaintain tumor cell growth and biosynthesis under aerobic conditions.Investigation on energy metabolism pathway in cancer cells has arousedthe interest of cancer researchers all around the world. In recentyears, plentiful studies suggest that targeting the peculiar cancerenergy metabolic pathways, including glycolysis, mitochondrialrespiration, amino acid metabolism, and fatty acid oxidation may be aneffective strategy to starve cancer cells by blocking essentialnutrients. Natural products (NPs) are considered as the “treasure troveof small molecules drugs” and have played an extremely remarkable rolein the discovery and development of anticancer drugs. And numerous NPshave been reported to act on cancer energy metabolism targets.Tomatidine is such a natural product whose employment in treatingvarious cancers and related cancer cachexia is part of various aspectsof the present invention.

Certain embodiments are directed to a method of treating bladder cancerin a subject in need of such treatment, such method comprisingadministering to a microbiome of a subject with bladder cancer aneffective amount of a bacterial composition comprising Bacilluscalmette-guerin, with the bacterial composition adapted to produce atleast one of tomatidine, p53 and rapamycin. Preferably, the bacterialcomposition comprises bacteria modified via a clustered regularlyinterspaced short palindromic repeats (CRISPR) CRISPR associated protein(Cas) system to express one or both of tomatidine and rapamycin, and inother embodiments, also p53. Certain embodiments are focused on treatingmetastatic bladder cancer. The microbiome employed may be the gut, oral,bladder or skin microbiome. Certain embodiments further includeemploying a microbe selected from the group consisting of Streptococcus,Actinomyces, Veillonella, Fusobacterium, Porphromonas, Prevotella,Treponema, Nisseria, Haemophilis, Eubacteria, Lactobacterium,Capnocytophaga, Eikenella, Leptotrichia, Peptostreptococcus,Staphylococcus, and Propionibacterium. One preferred embodiment involvesadministering a bacterial composition to the subject so that at least0.1 mg of rapamycin is provided to the subject each day. Preferably, thebacterial composition is modified via a CRISPR-Cas system to express oneof rapamycin and/or tomatidine, with preferred bacterial compositionsincluding one of a Chlamydia, Shigella flexneri, Mycoplasma bacteria,and H. pylori. In other preferred embodiments, the method comprisesadministering to a microbiome of a subject with bladder cancer aneffective amount of a bacterial composition comprising a bacteria thathas been modified to express a therapeutically effective amount oftomatidine and rapamycin, with the bacteria selected from the groupconsisting of Streptococcus, Actinomyces, Veillonella, Fusobacterium,Porphromonas, Prevotella, Treponema, Nisseria, Haemophilis, Eubacteria,Lactobacterium, Capnocytophaga, Eikenella, Leptotrichia,Peptostreptococcus, Staphylococcus, Propionibacterium, Chlamydia,Shigella flexneri, Mycoplasma bacteria, H. pylori, and Streptomyceshygroscopicus. The bacteria employed may be of a species found in thesubject's gut microbiome and may further have been modified using aCRISPR-Cas system to produce one of tomatidine or rapamycin. Atherapeutically effective amount of a bacterial composition may alsoinclude Streptomyces hygroscopicus in an amount effective to provide atherapeutically effective amount of rapamycin to the subject. Inparticular embodiments, especially directed to addressing bladdercancer, the bacterial composition comprises Bacillus calmette-guerin,and even more preferably, where the Bacillus calmette-guerin alsoproduces at least one of p53, rapamycin or tomatidine, and especiallywhere the method maintains or increases the muscle mass of the subject.As described in more detail in the detailed description of variousembodiments, still other agents, such as methylene blue, metformin,resveratrol (3,4′,5-trihydroxystilbene; C.sub.14H.sub.12O.sub.3), p53protein, spermidine, diallyl trisulfide, apigenin, cyclopamine,sulforaphane, curcumin and glucosamine are employed via the productionby microbes of an individual's microbiome to achieve the objective ofdelaying aging, and thus, in delaying and treating the onset of cancers.

While specific embodiments and applications of the present inventionhave been illustrated and described, it is to be understood that theinvention is not limited to the precise configuration and componentsdisclosed herein. Various modifications, changes, and variations whichwill be apparent to those skilled in the art may be made in thearrangement, operation, and details of the methods and systems of thepresent invention disclosed herein without departing from the spirit andscope of the invention. It is important, therefore, that the claims beregarded as including any such equivalent construction insofar as theydo not depart from the spirit and scope of the present invention.

Checkpoint inhibition, namely PD1/PD-L1 pathway inhibition, has shownimpressive results in many tumor types. One aspect of the presentinvention relates to the provision of checkpoint inhibitors inconjunction with bacterial formulations modified to express p53 and/ortomatidine. As the immune system is critically involved in thedevelopment, structural nature and progression of certain cancers, aninflammatory environment is believed to be related to tumor development.Chronic inflammation occurs due to tumor environment stress and thetumor microenvironment resembles an inflammation site, with metastaticsites creating a cytokine milieu conducive to tumor growth. Inparticular embodiments of the present invention, controlling cytokinesis desired at particular sites of an individual's body, rather thansystemic control of cytokines. Cytokines of the TNF family regulate awide range of different immune defense mechanisms, both of the innateand the adaptive types. However, when acting in excess, they can causesignificant damage. The ligands of the TNF family are cell-boundtransmembrane proteins and thus exert their effects largely by affectingonly cells that are located adjacently to the ligand-producing cell.Selective suppression of the ligand producing cells in situations wherethe ligand plays a pathogenic role forms one aspect of variousembodiments of the present invention, such as where destruction of cellsproducing a cytokine may be preferable over mere attempts to achievedirect blocking of the function of the cytokine molecules. Destructionof cytokine-producing cells prevents further synthesis of the cytokinesand provides durable protection. Blocking circulating cytokines affectsthe whole body. Destruction of cytokine-producing cells, in contrast,may be restricted to a particular site in the body while maintainingbeneficial effects of the cytokine at other sites. Using the methods andsystems as described herein, the direct and local administration ofagents, such as p53, statins, tomatidine, rapamycin, etc. can beemployed to achieve the desired non-systemic administration of suchagents to tissues.

In some embodiments, methods further comprise administering to thesubject an immune checkpoint inhibitor via cells within an individual'smicrobiome. Use of CRISPR-Cas systems to modify desired bacteria orother microbes to produce desired amounts of such inhibitors is thus oneaspect of the preset invention. In some embodiments, the immunecheckpoint inhibitor is a protein or polypeptide that specifically bindsto an immune checkpoint protein. In some embodiments, the immunecheckpoint protein is selected from the group consisting of CTLA4, PD-1,PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA. Insome embodiments, the polypeptide or protein is an antibody orantigen-binding fragment thereof. In some embodiments, the immunecheckpoint inhibitor is an interfering nucleic acid molecule. In someembodiments, the interfering nucleic acid molecule is an siRNA molecule,an shRNA molecule or an antisense RNA molecule. In some embodiments, theimmune checkpoint inhibitor is selected from the group consisting ofnivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110,TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475, CT O11, MPDL3280A,MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010. In some embodiments, theimmune checkpoint inhibitor is administered before the bacterialformulation. In some embodiments, the immune checkpoint inhibitor isadministered at least one day before the bacterial formulation. In someembodiments, the immune checkpoint is administered at about the sametime as the bacterial formulation. In some embodiments, the immunecheckpoint inhibitor is administered on the same day as the bacterialformulation. In some embodiments, the immune checkpoint inhibitor isadministered after the bacterial formulation. In some embodiments, theimmune checkpoint inhibitor is administered at least one day after thebacterial formulation. In some embodiments, the immune checkpointinhibitor is administered by injection. In some embodiments, theinjection is an intravenous, intramuscular, intratumoral or subcutaneousinjection.

Therefore, in some embodiments, the invention is directed to a systemand method of treating cancer in a human subject comprisingadministering to the subject an immune checkpoint inhibitor via theexpression thereof by an individual's microbiome, and includes, forexample, expression using bacteria of the genera Bifidobacterium. UsingCRISPR-Cas systems, one is able to achieve expression of genes and geneproducts in prokaryotic cells that provide desired amounts of checkpointinhibitors to a person so as to effectively treat various forms ofcancer. In such a manner, aspects of the present invention takeadvantage of the commensal relationship between the human host and themicrobiome for the targeted delivery of nucleic acid therapies. Incertain embodiments, employing the methods set forth herein one is ableto deliver nucleic acids to program bacteria for expression oftherapeutic proteins and RNA molecules in vivo at sites of greatestsignificance for a particular disease, thus providing for higher localconcentrations of therapeutic products while reducing off-targeteffects.

One aspect of the present invention is the targeting of the gutmicrobiota-dependent trimethylamine-N-oxide (TMAO) formation as atherapeutic strategy to reduce thrombotic risk. One aspect of variousembodiments is therefore to “drug the microbiome” for clinical purposes,including the maintenance of cardiovascular health. In one embodiment,choline analog inhibitors are selectively transported into gut microbes,thus limiting systemic drug exposure in the host. Choline accumulationis sensed as nutrient overload within gut microbes and promotes theinduction of the cut gene cluster, encoding CutC/D itself as well as acholine transporter As a result, a positive feedback loop isestablished, whereby both the choline TMA lyase substrate (choline) andsubstrate analog (the drug inhibitor) are actively pumped andsequestered into the microbe. In turn, this event reduces cholineavailability to neighboring microbes, further contributing as asecondary mechanism to the reduction of TMA formation. The suppressionof TMAO levels by choline TMA lyase inhibitors suppresses clot formationand provides for a potent antithrombotic effect of these compounds.Importantly, bleeding was not observed upon administration of the drugs,which represents a key and uncommon advantage for their as antiplatelettherapy. Modification of the gut microbiota composition to trigger ashift in the proportions of microbial communities such that an increasein the Akkermansia genus is observed that is believed to play aprotective role in obesity and metabolic health. Thus, various aspectsof the present invention include the shift of microbial composition toone that produces less TMAO and thus counteracts thrombotic risk, thuspreventing or treating diseases through microbiome targeting Modulatingthe microbiome can be achieved in different ways, ranging fromprobiotics and prebiotics to fecal microbiome transplants, thus, the useof bacteria as drugs can be seen as an effective way to treat variousdiseases.

To comply with written description and enablement requirements, allreferences cited herein, including but not limited to published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.Incorporated herein by this reference are the following US patentpublications: 20170079947 to Richards; 20140296139 to Cohen et al.;20160175327 to Adams et. al.; 20100081681 to Blagosklonny and20120283269 to Blagosklonny; U.S. Patent Publication Nos. 20140030332 toBaron, et al., 20070123448 to Kaplan et al.; 20160000841 to Yamamoto, etal.; 20160095316 to Goodman et al.; 20160158294 to Von Maltzahn;20140294915 to Kovarik; U.S. Pat. No. 8,034,601 to Boileau et al.;20130225440 to Freidman, et al., 20150071957 to Kelly et al.,20160151428 to Bryann et al.; 20160199424 to Berry et al.; 20160069921to Holmes, et al.; 20160000754 to Stamets; U.S. Pat. No. 9,044,420 toDubensky, Jr, et al.; 20160120915 to Blaser et. al.; 2014/0349405 toSontheimer; 2014/0377278 to Elinav; 2014/0045744 to Gordon; 2013/0259834to Klaenhammer; 2013/0157876 to Lynch; 2012/0276143 to O'Mahony;2015/0064138 to Lu; 2009/0205083 to Gupta et al.; 201/50132263 to Liu;and 2014/0068797 to Doudna; 2014/0255351 to Berstad et al.; 2015/0086581to Li; PCT/US2014/036849 and WO 2013026000 to Bryann; U.S. Pat.Publication No. 2015/0190435 to Henn; 2012/0142548 to Corsi et al.; U.S.Pat. Nos. 6,287,610, 6,569,474, U.S. 2002/0009520, U.S. 2003/0206995,U.S. 2007/0054008; and U.S. Pat. No. 8,349,313 to Smith; U.S. Pat. No.9,011,834 to McKenzie; 20150004130 to Faber et. al, 20160206666 to Falb;20160206668 to Kort et al; and WO2015069682A2 to Asesvelt, et. al.;20160199424 to Berry et al.; 20130326645 to Cost et al.; 2012/0276149 toLittman; U.S. Pat. No. 9,314,489 to Kelly et al.; U.S. Pat. PublicationNo. 2016/0024510 to Bikard et al.; U.S. Pat. Publication No.2018/0015131 to Gajewski et al.; U.S. Pat. Publication No. 2018/0371405to Barrangous et al. and U.S. Pat. Publication No. 2018/0140698 to Clubeet al.

Other aspects of the present invention relate to the reduction of thelikelihood of, treatment and/or prevention of cancer by interrupting amicrobial carcinogenic pathway, and by enhancing an individual'ssurvival. Various embodiments of the present invention use microbiotamodifications to improve the efficacy of existing treatments, bymodifying a patient's microbiome to address the treatment and prolongedsurvival of cancer victims.

Preferably, the modified bacteria employed in the present invention areadministered orally to a patient in order to deliver the therapeuticdirectly to the site of inflammation in the gut. The advantage of thisapproach is that it avoids systemic administration of immunosuppressivedrugs and delivers the therapeutic directly to the gastrointestinaltract. The viability and stability of such modified bacteria is enhancedto support the production of such microbes of desired agents, and bydoing so, a method is provided that reduces gut inflammation, enhancesgut barrier function, and/or treats autoimmune disorders. Preferably,such modified bacteria are capable of producing therapeuticanti-inflammation and/or gut barrier enhancer molecules, particularly inthe presence of reactive nitrogen species, and more preferably thebacteria are functionally silent until they reach an environmentcontaining local reactive nitrogen species (RNS), wherein expression ofthe therapeutic molecule is induced. In certain embodiments, thegenetically engineered bacteria are non-pathogenic and may be introducedinto the gut in order to reduce gut inflammation and/or enhance gutbarrier function. For example, in some embodiments, the bacteria areunder the control of a RNS-responsive regulatory region and acorresponding RNS-sensing transcription factor such that a desiredproduct, e.g. butyrate is produced, which induces the differentiation ofregulatory T cells in the gut and/or promotes the barrier function ofcolonic epithelial cells. Use of such modified bacteria, especiallythose modified via CRISPR-cas systems, provides a way to generate adesired therapeutic effect in a manner that lowers the safety issuesassociated with systemic exposure.

Various embodiments of the present invention are directed to the fieldof Oncology, and in particular, embodiments directed to a method ofameliorating, treating, or preventing a malignancy in a human subjectwherein the steps of the method assist or boost the immune system ineradicating cancerous cells. In certain embodiments, the administrationof beneficial bacteria to an individual's microbiome that have beenmodified so as to produce effective amounts of desired compositions,compounds, agents, etc, is done to address cancerous conditions. Inseveral embodiments, the administration of such beneficial bacteria andmicrobes to an individual's microbiome invokes either an active (or apassive) immune response to destroy, weaken or render less invasivecertain cancerous cells. Various other embodiments are drawn to theco-administration of biological adjuvants (e.g., interleukins,cytokines, Bacillus Comette-Guerin, monophosphoryl lipid A, etc.) incombination with conventional therapies for treating cancer. Inparticular, the co-administration of various pre-biotic compositions toenhance and sustain the desired effects of the beneficial modifiedbacteria forms another aspect of the present invention. In this regard,incorporation by reference of U.S. Patent publication No. 20160213702 toMaltzahn et al. is included as part of the written description ofvarious aspects of the present invention. For example, in view of thefact that the microbiota of humans is complex and varies by individualdepending on genetics, age, sex, stress, nutrition and diet, modifyingthe numbers and species of gut, oral, vaginal and skin microbiota canalter community function and interaction with the host. A number ofprobiotic bacteria known in the art, as well as some foods considered tobe ‘prebiotic’ that contain substances that promote the growth ofcertain bacteria and that stimulate beneficial microbiota shifts toimprove human health, can be employed in concert with the modifiedbacteria as described herein to effect desired cancer treatmentregimens. For example, the administration of glycans in an amounteffective to modulate the abundance of the bacterial taxa can be used toachieve better outcomes for cancer patients.

One application of the present invention is to provide a CRISPR-Casmodified bacteria, such as a lactobacteria, to a person diagnosed withcancer, so as to facilitate the production of agents in a manner that iseffective to preserve muscle mass and function in such individual. Otherembodiments include CRISPR-Cas modified bacteria that express levels oftumor suppressor factors, such as p53, in a manner that provides aneffective, therapeutic amount to an individual via the production ofsuch factors by one or more of the individual's microbiome (e.g. gut,oral, skin, vaginal, etc.) By having the individual's microbiomeresponsible for administration of such factors, instead of attempting toadminister such factors via more traditional routes, such as injection,pills, etc., it is believed that a better result can be attained in amuch more natural fashion.

Moreover, in view of the ability to further modify bacteria in variousways to provide desired factors at particular times, or in conjunctionwith particular agents, it is possible to fine tune the administrationof desired factors, such as p53, so as to reduce any under or overproduction thereof. For example, rendering particular modified bacteriasensitive to a predetermined antibiotic can thus provide a way to reducethe numbers of any given modified bacteria in a manner to control thepopulations of such bacteria in an individual's microbiome, and hence,control the level of production of factors produced by such bacteria. Tocomply with written description and enablement requirements,incorporated herein by the following references are the following patentpublications: U.S. Patent publication Nos. 20140349405 to Sontheimer;20140377278 to Elinav; 20140045744 to Gordon; 20130259834 toKlaenhammer; 20130157876 to Lynch; 20120276143 to O'Mahony; 20150064138to Lu; 20090205083 to Gupta et al.; 20150132263 to Liu; and 20140068797to Doudna; 20140255351 to Berstad et al.; 20150086581 to Li;PCT/US2014/036849 and WO 2013026000 to Bryan; 20160199424 to Berry etal.; 20130326645 to Cost et al.

CRISPR-based genetic editing tools offer an efficient way to manipulateexpression levels of multiple genes and to provide a solution towardsthe “multivariate modular metabolic engineering” to optimize the drugsynthesis pathways with modular, multiplex regulation using only a fewcore proteins (e.g., dCas9) that are guided to specific sequences byguide RNAs.

In still other embodiments of the present invention, modifying bacteriaso as to administer them to a person's microbiome is performed in amanner so that particular agents, factors or proteins derived frommushrooms, are rendered possible, with desired mushroom derivedcomponents believed to have anti-cancer characteristics, either alone orwhen used in conjunction with other agents. In particular, by assessinginitially the particular bacterial constituents of an individual'smicrobiome and then administering to such individual a similar speciesof microbe, but one which has been modified, preferably via employmentof a CRISPR-Cas system, one is able to effectively administer to suchindividual various desired anti-cancer treatments in a way that isbelieved to be far less disruptive, efficient and dependable as comparedto other routes of administration. The modification of speciallydesigned bacteria that reside in a person's body is believed toalleviate the concerns regarding genetic alteration of the human genome,as what is being modified is a microbiome that is present in a person'sbody—but is not directly involved in the human genome itself. There area myriad of ways to combine various triggering factors to turn on or offparticular productions of agents, factors or proteins that may beincluded in such modified microbiome species. The present invention invarious embodiments is directed to at least those embodiments wherecancer therapeutic agents can be administered by the microbiome of theindividual that has cancer so as to effectively treat the cancer and/orremedy the symptoms resulting from the disease.

One aspect of the present invention is directed to the employment andmodification of an individual's microbiome to address muscle massretention and as a corollary thereof, to address the counterpart ofobesity by lessening the amount of fat storage by such individual. Incertain embodiments, the provision of effective amounts of tomatidine isrendered available to an individual. Still other embodiments alsoinvolve the reduction in the amount of acetate levels in an individual'sbody, which in turn lowers the amount of insulin the individual willproduce, which has the effect of keeping fat cells from storing moreenergy in the form of fat. The reductions in the amount of acetateavailable in an individual's body further reduces the amount of thehormone ghrelin, thus reducing the hunger drive of the individual. Thus,the modification of an individual's microbiome influences variousaspects of their metabolism in a manner that not only retains andmaintains the ability to nurture muscle tissue, but to also reduceobesity by affecting the amount of fat that the body stores. While notbound by theory, it is believed that the gut bacteria of an individualis a substantial source of acetate production. The production of acetateby gut microbes is believed to send signals to the brain of theindividual to initiate the production of insulin, conveyed via the vagusnerve. Fine tuning of the amount and type of gut microbes (e.g. via theuse of antibiotics to initially reduce the kind and numbers of undesiredbacteria, followed by purposeful inoculation of an individual's gutmicrobiome with modified microbes, e.g. via CRISPR-Cas insertion ofparticular factors, proteins, etc.) is an effective way to address notonly muscle wasting issues, but also obesity issues of individuals.

While there are many gut bacteria that produce acetate, particularbacteria are preferably selected and even more preferably are modifiedusing CRISPR-Cas systems to address the levels of acetate productiononce such bacteria are introduced to an individuals' microbiome.Preferably the gut microbiota are members of two bacterial divisions:the Bacteroidetes and the Firmicutes. The modification of anindividual's gut microbiome is directed in a manner such that thetypical increase seen in the relative abundance of the Firmicutes and acorresponding division-wide decrease in the relative abundance of theBacteroidetes in obese individuals, is addressed. Obese people have moreFirmicutes and almost 90% less Bacteroidetes than the lean people.Preferably, the administration of modified Bacteroidetes is achieved tomore substantially reflect gut populations in more lean individuals, andby doing so, reducing the amount of acetate produced by the overall gutmicrobiome. Such a shift in the population of gut microbes to favorBacteroidetes over Firmicutes, whether or not coupled with theadministration of tomatidine, is one aspect of the present invention'sobjective of achieving a greater proportion of muscle mass than fat thatwould otherwise occur in any given individual. In still otherembodiments, addressing the acetate production by especially Firmicutes,which has an increased capacity for fermenting polysaccharides relativeto the lean-associated microbiome, is another way to achieve thisobjective, and addresses the significant obesity issues especiallyprevalent in Western societies.

In yet another embodiment, bioadhesive strips are provided that haveencapsulated structures are filled with desired agents, including butnot limited to tomatidine and/or microbes, especially bacteria that arefound in an individual's oral microbiome, such that effective amounts ofthe agents can be administered to treat particular diseases includingmuscle atrophy. Preferably, the bacteria comprise bacteria that arefound in the communities of healthy mouths, including, for example,Streptococcus, Actinomyces, Veillonella, Fusobacterium, Porphromonas,Prevotella, Treponema, Neisseria, Haemophilus, Eubacteria,Lactobacterium, Capnocytophaga, Eikenella, Leptotrichia,Peptostreptococcus, Staphylococcus, and Propionibacterium. Such stripsmay be manufactured to have desired dissolvable aspects thereto and thatfurther have encapsulated portions that house desired agents, such asbut not limited to tomatidine, p53 protein, and other agents able totreat cancer symptoms.

Some bacterial pathogens actively inhibit p53 protein and induce itsdegradation, resulting in alteration of cellular stress responses. Forexample, in gastric epithelial cells infected with Helicobacter pylori,a bacterial pathogen that commonly infects the human stomach, gastriccancer is more common. In addition to H. pylori, a number of otherbacterial species also inhibit p53, providing further evidence thathost-bacteria interactions reveal that bacterial infections areassociated with tumorigenesis. Inhibition of p53 may provide certainbenefits to bacteria, for example, it is believed that the inhibition ofp53 may allow bacteria to subvert the host cell cycle control andapoptosis mechanisms, resulting in inhibition of cell death and survivalof host cells damaged by infection.

In certain embodiments, CRISPR-Cas systems are employed to interferewith the p53 degradation abilities of particular bacteria that are knownto degrade or otherwise interfere with the ability of p53 to function.As such in certain embodiments, the bacterial species is selected fromthe group consisting of a Chlamydia species, Shigella flexneri,Mycoplasma bacteria, and H. pylori.

The methods described herein are useful for treating and/or preventing(i.e., reducing the likelihood or risk of occurrence) differentdiseases, disorders, and conditions such as cancers and infectiousdiseases. Cancer remains the second most frequent cause of death inindustrialized societies. Conventional therapies like surgery,radiotherapy, or chemotherapy remain the backbone of cancer therapy todate. Since the late 1980s, oncologists were successfully using thevaccine variant of Mycobacterium bovis BCG (Bacille Calmette-Guerin) asagent to prevent relapses of bladder cancer after surgical removal ofthe primary tumor. Although the exact mode of action of the bacteria isnot fully understood, they might enhance the immune response against thecancer cells by, for example, activation of natural killer cells.

All references cited herein, including but not limited to published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.Incorporated herein by this reference are the following US patentpublications: US Patent Publication Nos. 20140030332 to Baron, et al.,and 20070123448 to Kaplan et al.; 20160000841 to Yamamoto, et al.;20160095316 to Goodman et al.; 20160158294 to Von Maltzahn; 20140294915to Kovarik; U.S. Pat. No. 8,034,601 to Boileau et al.; 20130225440 toFreidman, et al., 20150071957 to Kelly et al., 20160151428 to Bryann etal.; 20160199424 to Berry et al.; 20160069921 to Holmes, et al.;20160000754 to Stamets; and U.S. Pat. No. 9,044,420 to Dubensky, Jr, etal.

While the provision of microbes, preferably bacteria, to a personsuffering from cancer is via their gut microbiome, other microbiomes maybe employed, e.g. other than the microbes that colonize thegastrointestinal (GI) tract, as there exist microbiomes on the skin, andin other epithelial and tissue niches such as the oral cavity, nasalpassages, eye surface and vagina. Each of these microbiomes may betargeted for delivery of therapeutic agents to address cancer issues,including muscle atrophy associated with cancer. The gastrointestinaltract (as well as the other mentioned microbiomes) harbors an abundantand diverse microbial community, including diverse strains of bacteria.Hundreds of different species may form a commensal community in the GItract in a healthy person, and this complement of organisms evolves fromthe time of birth to ultimately form a functionally mature microbialpopulation. A healthy microbiota provides the host with multiplebenefits, including colonization resistance to a broad spectrum ofpathogens, essential nutrient biosynthesis and absorption, and immunestimulation that maintains a healthy gut epithelium and an appropriatelycontrolled systemic immunity.

In one embodiment of the present invention, a method and system andcomposition is provided to populate a person's microbiome, preferablytheir oral or gut microbiome, to restore, maintain or promote health ofthe individual and/or to alter a dysbiosis. For example, periodontaldisease, a common chronic inflammatory disorder, has been associatedwith increased risk of postmenopausal breast cancer, particularly amongformer smokers who quit in the past 20 years. There is a need to designmicrobial compositions so that they possess a plurality of beneficialproperties that would enhance the utility and commercialization of amicrobial composition, especially those modified to produce desiredagents, such as tomatidine. The human gut microbiota contains more than500-1000 different phylotypes belonging essentially to two majorbacterial divisions, the Bacteroidetes and the Firmicutes. The enhancedmetabolic activities of the colonized gut ensure that otherwiseindigestible dietary components are degraded with release of byproductsproviding an important nutrient source for the host. Similarly, theimmunological importance of the gut microbiota is well-recognized and isexemplified in germfree animals which have an impaired immune systemthat is functionally reconstituted following the introduction ofcommensal bacteria. T cell development and differentiation may requirecolonization by specific commensal micro-organisms.

In yet further aspects of the present invention, the treatment of cancerand cancer cachexia can be addressed by administering to an individual adesired amount of a bacteria modified (preferably via CRISPR-Cassystems) to produce predetermined levels of tomatidine and/or p53protein. Inclusion of DNA in microbes to produce certain p53 protein ina person's microbiome is one way in which to administer amounts to aperson's body in a manner that can be tolerized to such amounts.Effective ways to ensure that the type, amount, and timing of desiredadministration is achieved is made possible by the ability to promptlyaddress the survival of these modified microbiomes by use ofantibiotics, or any number of other ways to either increase or decreasethe efficiency of such modified microbes. Such a system and methodaddresses concerns with respect to proper timing of amounts that theperson is being properly exposed to due to effective ways to control theadministration of such factors, proteins, drugs, etc. via one'smicrobiome.

The particular protein degradation pathway which seems to be responsiblefor much of the muscle loss seen in a muscle undergoing atrophy is theATP-dependent, ubiquitin/proteasome pathway. In this system, particularproteins are targeted for destruction by the ligation of at least fourcopies of a small peptide called ubiquitin onto a substrate protein. Inskeletal muscle, the E3 ubiquitin ligases atrogin-1 and MuRF1 are knownto play essential roles protein degradation and muscle atrophy.

The bone is a common site of metastasis for several malignancies. Theimpact of metastasized tumor cells in the bone disrupts the balancebetween the activities of the osteoclasts and osteoblasts.Radiographically, bone lesions are classified as being osteolytic (boneloss) or osteosclerotic (bone formation) or mixed.

Osteopontin is expressed by tumor cells and stimulates osteoblastdifferentiation. Osteopontin is a pro-inflammatory cytokine and isimplicated in the progression of liver tumors, as well as other tumors.Tumor cells with a competent Hedgehog pathway are more potent atinducing osteoblast differentiation. The Hedgehog pathway plays anessential function in regulating cell fate and in developmentalpatterning in humans and is important in the formation of the skeleton.During skeletogenesis and endochondral ossification, Hedgehog pathwaysignaling coordinates growth and differentiation. Reactivation of theHedgehog pathway has been implicated in a wide variety of cancers andcarcinogenesis.

Tumor cells initially enhance the differentiation of osteoblasts that inturn, express osteoclastogenesis enhancing factors. Later, as theosteoblasts get eliminated, an environment is created that stimulatesosteoclast differentiation and activity. Thus, an active Hedgehogpathway signaling in the tumor cells facilitates the generation of anosteoclast-stimulating milieu by initially kick starting osteoblastdevelopment. Tumor cells can alter the balance between the activities ofosteoblasts and osteoclasts via Hedgehog pathway signaling. For example,breast cancer cells express Hedgehog pathway ligands and the Hedgehogpathway signaling propels breast cancer progression. Administration ofpharmacological Hedgehog pathway inhibitors can inhibit Hedgehog pathwaysignaling in breast cancer cells and Hedgehog pathway proteins and geneshave also been implicated in esophageal cancer, stomach cancer, prostatecancer, ovarian cancer, biliary tract cancer, glial cell cancer,multiple myeloma, colon cancer and melanoma. While not bound by theory,tomatidine is believed to interfere with the Hedgehog pathway and mayinhibit the actions of the pro-inflammatory cytokine Osteopontin.

Hedgehog pathway signaling in the tumor cells is essential to thedevelopment of osteolytic metastases. Soluble factors, that includeOsteopontin (OPN) and other Hedgehog pathway ligands, are secreted bytumor cells, and are thought to enhance osteoblast differentiation andmineralization activity. Tumor cells initiate osteoblast differentiationand the expression of osteoclastogenic factors is seen as an earlyevent, followed by elimination of osteoblasts later. Thus, the overallmicroenvironment in cancer progression appears to shift in favor ofosteoclastogenesis. An active Hedgehog pathway signaling and expressionof OPN are important attributes for a tumor cell to activate osteoclastdifferentiation and resorptive activity.

Blocking the Hedgehog pathway interferes with the transcription ofOsteopontin (OPN). Cyclopamine treatment decreases the activity of theOPN promoter in a dose-dependent manner. In contrast, tomatidine, thestructural analog of cyclopamine, has apparently no effect on thepromoter activity of cyclopamine. This is surprising but reveals thatthe particular mode of action of tomatidine in the body is as yet notfully known.

Tumors are now recognized as comprising of a mosaic of geneticallydifferent and actively mutating cells rather than a single type. Thus,combination drug therapies are being advocated to combat tumor cellularheterogenecity. Employing various methods of the present invention, itis believed that such combination therapies can be achieved by using anindividual's own microbiomes, such that one or more drugs, factors,proteins, (e.g. tomatidine and p53) can be provided to an individualthrough the modification of the individual's microbiome.

In spite of great advances in understanding pathways related to cancerand cancer therapy, there is a need to provide new anticancer treatmentsthat do not cause toxicity to healthy cells and that is effective intreating cancer, and especially the cachexia associated therewith. Thepresent invention provides such a treatment option, and one that is inmany ways, more subject to the inherent control aspects of the humanbody as it relies upon the long established but poorly understoodrelationship between diseases and the microbiomes of humans.

In still other embodiments of the present invention, embodiments relateto the employment of anti-muscle atrophy characteristics of tomatidineas described herein, but further involve the employment and productionof anticancer proteins via the microbiome of an individual, e.g. thatare capable of oral administration, and are preferably stable at roomtemperature, and in some embodiments also have potent antiviralactivities that can be useful in a significant percentage of humancancers that are caused by viral infections. One aspect of the presentinvention is directed to the use of CRISPR-Cas systems to provide abetter p53 protein that is much more stable so that its folding ispreserved, thus protecting its tumor suppressing function. Inparticular, the regions where the protein are most vulnerable tomutations that cause improper folding are targeted and revised so as toimpede such mis-folding events.

The microbiota inhabiting our bodies influence cancer predisposition andetiology. The largest microbial community in the human body resides inthe gut and comprises somewhere between 300 and 1000 different microbialspecies. The human oral microbiome and the bacteria inhabiting suchmicrobiome are, in certain circumstances, also effective as agents inthe treatment of cancer. Various embodiments of the present inventioninvolve the modification of at least two, if not three separatemicrobiomes of a person to treat certain conditions. For example, thetreatment for cachexia may be achieved via modification of anindividual's oral microbiome via the delivery of particular bacteriadesigned to produce therapeutic amounts of tomatidine. The simultaneousprovision of bacteria to the individual's gut microbiome that aredesigned to produce therapeutic amounts of p53 protein can also beachieved, with the two separate microbiomes being employed to addressseparate but related issues involved in cancer treatments. Thisparticular aspect of the present invention, while simple in nature, isbelieved to have profound effects in avoiding undesired druginteractions that can complicate treatment regimens. By having differentmicrobiomes of the same individual administer different desiredcompounds, drugs, factors, proteins, etc. to the person's body, theability to separately control administration and amounts (as well as toaddress issues by killing bacteria in one but not the other microbiome)is rendered feasible as a way to administer desired cancer fightingagents to an individual.

Still other aspects of the present invention are directed to the use ofantibodies against certain oral bacteria, e.g. P. gingivalis—which is apathogen known to contribute to periodontal disease and that has beenassociated with the risk of pancreatic cancer, and lung cancer,colorectal cancer and cancers of the stomach, oesophagus, and cancers ofthe head and neck. Microbes mediate the relationships with these typesof cancer and epidemiologic evidence exists that shows the associationbetween the human microbiome and these types of cancer. Particularembodiments relate to an association of the oral microbiome andperiodontitis with pancreatic cancer, with a strong positive associationbeing noted between periodontitis at baseline and subsequent risk offatal pancreatic cancer. Men with periodontal disease have a 64% higherrisk of pancreatic cancer compared to those reporting no periodontaldisease and have a 4-fold increase in risk of pancreatic cancer.Elevated antibodies to P. gingivalis have been associated with a 3-foldincrease risk of aerodigestive cancer mortality.

One aspect of the present invention is directed to the use of commensaland symbiotic microbiota that have tumor-suppressive properties. It isbelieved by the present inventors that the associations between diet andcancer risk is explained by differences in microbiota among theparticipants and that the employment of probiotics and prebiotics is aneffective chemoprevention strategy that can be utilized to promotehealth and cancer treatment and recovery. Enhancing the microbiota of anindividual with particular microbes, such as those modified byCRISPR-Cas systems, to include, for example, the provision of one of p53protein expression or tomatidine expression, is one of the aspects ofvarious embodiments of the present invention.

While cancer has been largely perceived as an intrinsic problem of bodyhomeostasis, and infection a problem of external environment, thepresent inventors believe that the effective treatment of both diseasesconverges in that premalignant cell behavior is a mirror of cellulardysbyosis. Infection, like cancer, is a lack of regulation of importantcells of the superorganism of the human body in concert with itsintegral microbiomes. There appears to be a strong association betweenthe human microenvironment, sustained inflammation, and cancer. Growingevidence has emerged that, for example, the oral microbiome andperiodontitis has a profound impact with respect to the pathogenesis andrisk of various malignancies.

One aspect of the present invention relates to a paradigm shift from theclassic germ theory so prevalent in western medicine during the lastcentury. Viewing the microbiome of a person as an integral part of thehuman person in terms of health, much as an organ of such individual, isa more correct and useful concept as it relates to understanding cancerand in treating the same. Just as methods for addressing microbe baseddiseases has advanced recently, the ability to address cancer treatmentsfrom a new perspective is critical in advancing effective treatments toavoid if not cure various cancers. There are parallels between infectionby microbes and cancer from various perspectives. For example, a singleinfection can arise from a single microbe, just like a cancer can beinitiated by a single cell, and then spread to establish tumors andmetastatic disease. In both cases, disruption of homeostasis allows forpathological bacterial expansion and may lead to full blown infection.But not all pathologic microbes lead inevitably to infection, as such acourse is arrested by immune system responses. Similarly, precancerouscells and tissues do not always progress to full blown cancer, butrather, the progression of cancer is hindered or halted by the immunesystem. Premalignant cell behavior is a virtual mirror to microbialdysbiosis. Cancer, like infection, can be viewed as a dysbiosis of aperson's microbiome.

Despite the success of colonoscopy screening, colorectal cancer remainsone of the most common and deadly cancers, and colorectal cancerincidence is rising in some countries where screening is not routine andpopulations have recently switched from traditional diets to westerndiets. Colorectal cancer represents an important disease as one of themajor causes of death worldwide.

More than 50,000 people are diagnosed with pancreatic cancer every year,and because the disease is often not diagnosed until an advanced stage,less than 10% of those diagnosed will still be alive in five years.People with two types of periodontal disease have a greater risk ofsubsequently developing pancreatic cancer, showing that markers ofpancreatic cancer can be found in oral microbiome dysbyosis. Oralbacteria is the underlying explanation due to periodontitis being causedby oral bacteria dysbiosis. For example, individuals who havePorphyromonas gingivalis in their oral microbiome have an almost 60%greater risk of developing pancreatic cancer relative to those who donot have such bacteria in their oral microbiome. Similarly, individualswith Aggregatibacter actinomycetemcomitans present in their oralmicrobiome have at least a 50% increased relative risk of developingpancreatic cancer. Thus one aspect of the present invention is directedto the establishment of and maintenance of an oral microbiome of anindividual such that there are less robust populations of at least oneof Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans ina person's oral microbiome. One of the major challenges to detectingpancreatic cancer is its late clinical presentation. By the timepancreatic cancer is diagnosed, the cancer is usually well-advanced.Pancreatic adenocarcinoma is a low-incident but highly mortal disease.It accounts for only 3% of estimated new cancer cases each year but iscurrently the fourth common cause of cancer mortality. By 2030, it isexpected to be the 2.sup.nd leading cause of cancer death.

In one aspect of the present invention, a method includes the bacterialanalysis of an individual's stool in a non-invasive way for screeningfor pancreatic cancer. Preferably, a non-invasive, stool-based screeningtool for colorectal cancer is employed and a kit is used such thatpatients can use and send the kit in via mail for evaluation.

Throughout evolution, bacteria progressively acquired virulence factorsand the disease-promoting and pro-carcinogenic effects of pathogensdepend on these virulence factors, which often comprise adhesionmolecules, which confer the ability to adhere to and invade the tissuesof the human body. One aspect of the present invention is directed tothe modification of such adhesion molecules so that pathogenic bacteriaare altered in a manner that reduces their abilities to adhere totissues of the human body, thus lessening various human diseases.

Chronic and/or excessive consumption of alcohol has been found to be animportant risk factor for many cancers, including colorectal cancer.Microbial metabolism may contribute to the toxicity of alcohol,especially in the gastrointestinal tract, where aerobic and facultativeanaerobic bacteria convert ethanol to acetaldehyde. Indeed, acetaldehydeis known to be a highly toxic and pro-carcinogenic compound with variousnegative effects, ranging from DNA damage and impaired DNA excisionrepair to the degradation of folate. Thus one aspect of variousembodiments of the present invention is directed to providing particularbacteria to a person who consumes alcohol in a manner that lessens therisk of cancer via the ability of such bacteria to ameliorate theaccumulation of acetaldehyde. The conversion of ethanol to acetaldehydeis inhibited by the use of antibiotics, such as ciprofloxacin, whichkills primarily aerobic and facultative anaerobic bacterial populations.Thus, to reduce the undesired effects of alcohol conversion toacetaldehyde, the use of specific antibiotics, followed by the use ofprobiotics and/or fecal transplantation protocols, is one aspect of thepresent invention that may be employed to combat colorectalcancer-associated dysbiosis and thus restore eubiosis in chronicdiseases, helping to reduce microbiota-induced genotoxicity andactivation of inflammatory, proliferative and pro-carcinogenic pathways.The gut microbiota plays a major role in the promotion and progressionof colorectal cancer via several mechanisms, including inflammation,metabolism and genotoxicity, and thus, targeting an individual'smicrobiota is an effective way to treat, if not prevent, colorectalcancer. Particular bacterial species have been identified that aresuspected to play a role in colorectal carcinogenesis, includingStreptococcus bovis, Helicobacter pylori, Bacteroides fragilis,Enterococcus faecalis, Clostridium septicum, Fusobacterium spp. andEscherichia coli. Cancer incidence is low in the Ohio Amish and it isbelieved by the present inventors that the presence of Prevotellabacteria as more predominant bacteria in both their oral and gutmicrobiomes, is related to such lower cancer incidence. The gutmicrobiota of various livestock species has been reported to contain ahigh relative abundance of the xylanolytic bacterial species Prevotella.The present inventors submit that the environment plays an importantrole in modulating bacterial community composition and that transmissionof gut microbes occurs across host species. Gut microbial communitiesoften contain many Bacteroides or their close relatives, Prevotella. Oneaspect of certain embodiments of the present invention is directed toincreasing the prevalence of Prevotella populations in individuals so asto lessen the chances of cancer developing in such individuals. Stillother embodiments are directed to the modification of Prevotellabacteria in a manner that makes them less virulent, but that stillmaintain the beneficial effects of such bacteria in various microbiomes,such as the oral and gut microbiomes, e.g. by reducing the expression ofvirulence factors of Prevotella.

Another aspect of the present invention is directed to expression ofparticular tumor suppression agents by microbes in an individual'smicrobiome. Among tumor suppressor agents, the p53 protein is atranscription factor that recognizes and binds to specific DNA responseelements and activates gene transcription. P53 is a tumor suppressorthat has a role in the maintenance of genomic integrity and as aguardian of DNA. p53 secretion and uptake by cells demonstrates that p53is a transmissible particle. Stress triggered by ionizing radiation orother mutagenic events leads to p53 phosphorylation and cell-cyclearrest, senescence, or programmed cell death. Tumor suppressors arecomplex macromolecules normally occurring as multi-domain proteinsflanked by disordered segments. The tumor-suppressor p53 is atranscriptional factor that exerts broad anti-proliferative effects,including growth arrest, apoptosis, and cell senescence after cellularstress, and has been described as the most frequently mutated gene incancer cell. The end regions of tumor suppressor p53 act as molecularantennas for proper activity and interactome signaling. Althoughclassified as a transcription factor, p53 can also mediate apoptosis.

Most human viruses impair p53 activity. For example, in cervical cancer,the human papillomavirus E6 protein targets p53 for degradation.Bacterial infection is known to trigger the p53 pathway and activatesp53 isoforms. Another aspect of the present invention is directed to theinvolvement of p53 aggregates in cancer pathogenesis and progression.The production of competent p53 by bacteria in a person's microbiome isa better way in which to provide sufficient amounts of p53 to suppresstumorgenesis. Thus, one aspect of the present invention relates to theuse of CRISPR-Cas to modify bacteria to express p53 proteins, andpreferably a more stable p53 protein in that its folding is preserved,thus protecting its tumor suppressing function. The regions where theprotein are most vulnerable to mutations that cause improper folding aretargeted and revised so as to impede common mis-folding events.

In yet other embodiments, cancer cells are infected with modifiedbacteria having thermosensors, thus making such cancer cells amenable tosome controls incorporated therein, such as by adjusting the temperatureto turn on certain genes—with such genes encoding toxins. Alternatively,one is able to shut off the genes by adjusting the temperature, thusselectively killing or modifying the behavior of the cells infected bythe bacteria.

The prokaryotic type II CRISPR-Cas9 (clustered regularly interspacedshort palindromic repeats-CRISPR-associated 9) system is rapidlyrevolutionizing the field of genetic engineering, allowing researchersto alter the genomes of a large range of organisms with relative ease.Experimental approaches based on this versatile technology have thepotential to transform the field of cancer genetics.

Yet another aspect of the present invention relates to an increased riskof pancreatic cancer in patients with Helicobacter pylori (H. pylori)and that is also dependent on particular blood types with an associationbetween pancreatic cancer risk and CagA-negative H. pyloriseropositivity found among individuals with non-O blood type, but notamong those with 0 blood type. The differences in terminal bindingantigens in gastrointestinal mucins for individuals with non-0 bloodtype (A and B), influences the binding potential of the H. pylori. Thereis therefore a link between oral disease and pancreatic cancer and thebacteria found in certain types-of gum disease is linked to a 2.times.greater risk of developing pancreatic cancer. Pancreatic cancer, whichis difficult to detect and kills most patients within six months ofdiagnosis, is responsible for 40,000 deaths a year in the United States.Antibodies for oral bacteria are indicators of pancreatic cancer risk.

MicroRNAs (miRNAs) are short, noncoding RNAs that regulate target mRNAsvia transcript degradation or translational repression. Cell- andtissue-specific miRNA expression profiles are altered in numerousdisease states. Inflammatory bowel diseases (IBD) are a major riskfactor for the development of colon cancer. The loss of all of theintestinal miRNA results in impaired barrier function and inflammationsimilar to IBD. Circulating miRNA profiles are known to correlate withmiRNA expression changes in diseased tissue. While conventional effortsto treat cancer have focused on the inhibition/destruction of tumorcells, strategies to modulate the host microbiota and miRNAs-inducedinflammation offer a new way by which to combat what has been a terriblydifficult disease to address. Antibiotic treatment causes disturbance ofthe microbiota, and probiotics, prebiotics and fecal microbiometransplantation may be employed to restore the dysbiosis caused thereby.An individual's microbiota is tied into certain cancers, includingcolorectal cancer, by induction of a chronic inflammatory state, leadingto the production of toxic metabolites. Microorganisms frequently foundin IBD patients include different species that are well known butyrateproducing bacteria, which are linked to disease severity. Thus, oneaspect of the present invention relates to the modification of anindividual's microbiota to reduce the amount of butyrate producingbacteria, or at least the amount of butyrate by the bacteria present inan individual's microbiota, especially their gut microbiome.

Molecular mechanisms modulated by gut microbiota promote inflammationand support colorectal carcinogenesis. Both endogenous and exogenousmiRNAs modulate tumor-related inflammation in colorectal cancer. Gutmicrobiota has an influence on colorectal carcinogenesis and the microbepopulation living in the human intestine plays a significant role in thedevelopment and progression of colorectal cancer. Maintenance of ahealthy intestinal epithelia is critical to provide optimal nutrientabsorption, as well as an efficient immune barrier. The balance betweenintestinal microbiota, intestinal epithelium and host immune system isdecisive for normal functionality of the intestinal cells. Therefore,changes in any of these three factors may influence the functionality ofthe intestinal epithelium. The benefits of the body in relation to gutmicrobiota are related to extraction of the energy from the fermentationof undigested carbohydrates and from the absorption of short-chain fattyacids. Butyrate is the most important of these fatty acids beingmetabolized by the colonic epithelium and is the favorite energy sourceof colonocytes. The most important bacteria producing this fatty acidare Faecalibacterium prausnitzii, which belongs to the Clostridiumleptum cluster, and Eubacterium rectale/Roseburia spp., which belong tothe Clostridium coccoides. In healthy colonocytes, butyrate hampersapoptosis and further mucosal atrophy. In colorectal cancer cells,butyrate has been proved to stimulate differentiation, impede cellproliferation, lead to apoptosis and inhibit angiogenesis.

Butyrate protects human colon cells from DNA damage. In addition tobutyrate, gut microbiota are also implicated in the constitution ofanother category of beneficial fatty acids, such as conjugated linoleicacids, having anti-inflammatory and cancer protective properties.

The composition of gut microbiota evolves throughout human life, frombirth to old age, and is modulated, temporarily or permanently, by manyfactors such as dietary components, environment, age, stress, treatment(medical or surgical) and disease. Antibiotic-based therapy representsone of the most important factors with the effect on the composition ofthe microbiota. This therapy can cause diarrhea which generally isassociated with altered intestinal microbiota resulting inenteropathogens overgrowth, loss of mucosal integrity and alteredmetabolism of vitamins and minerals. The elderly have significantlydifferent microbiota than younger adults.

Individuals can be classified into one of three prevalent variants or“enterotypes” according to the abundance of predominant genera which areBacteroides, Prevotella and Ruminococcus. Bacteroides enterotypes arerelated to amino acids, animal proteins and saturated fats, constituentstypical to Western diet, while Prevotella is connected to carbohydratesand simple sugars, suggesting an interconnection with acarbohydrate-based diet more common of rural societies.

Individuals whose microbiota are mainly Bacteroides and commute theirdietary patterns to a diet based on high proportions of carbohydrates,will acquire a Prevotella enterotype in the long term. Substantialchanges in the composition of fecal microbiota are detectable in a fewdays after carbohydrate intake, demonstrating that diet rapidly andreproducibly alters the human gut microbiome. Numerous studies indicatethat fruit, vegetable and a high-fiber intake, particularly of cerealsand whole grains, is associated with a decreased risk of colorectalcancer, while diets that are rich in red and processed meat, fat andalcohol are associated with an increased risk of the disease. Higherdietary intakes of animal products may modify gut microbiota andconsequently play an important role in carcinogenesis.

One aspect of the present invention is the modification of anindividual's gut microbiome such that they harbor far less of thebacteria Streptococcus bovis, S. bovis bacteremia, Clostridia,Bacteroides and Helicobacter pylori, all of which have been involved inthe pathogenesis of cancer.

Conversely, bacteria like LactoBacillus and Bifidobacterium haveanticarcinogenic effects, which are believed to involve inactivation ofmicrobial enzymes which are important for pro-carcinogen activation. L.casei and L. acidophilus decrease the activity of .beta.-glucuronidase,azoreductase, and reflect that the balance of activation anddetoxification supports the belief that the microbial communitystructure plays a significant role in the initiating step ofcarcinogenesis. One aspect of the present invention relates to thefavorable modulation of the gut microbiota structure to reduce the riskof cancer development e.g. by the clinical use of probiotic in theprevention of cancer. In various embodiments, the probioticsupplementation of an individual's microbiome is able to modifymicrobiota structure by reducing enterobacteria like Salmonella/Shigellaand increasing lactic acid bacteria and Bifidobacteria to provide aprotective role of such probiotics.

Inflammatory bowel diseases (IBD) are induced and preserved by diversemicroorganisms and frequently involves signs of global dysbiosis,according to changes in the number, diversity and stability ofmicrobiota. Increasing evidence shows that dysbiosis induces theproduction of genotoxins and metabolites associated with tumorigenesisand produces disorder of the immune response which promotes andmaintains inflammation in IBD leading to colorectal cancer.Microorganisms frequently found in IBD patients include differentspecies of E. coli, species of Chlamydia, Mycobacterium, Clostridia,Candida, as well as Proteus mirabilis, Klebsiella pneumonia and diverseProteobateria, including Helicobacter. Firmicutes and Bacteroidetesdecrease in IBD. Different bacterial species contribute to thepathogenesis of IBD, with enhanced activation of transcription factorNF-.kappa.B, an important regulator of inflammatory processes.NF-.kappa.B suppression improves IBD development, and NF-.kappa.Bdependent cytokines are key agents which signal from inflammatory cellsto tumor cells. In chronic inflammation, proinflammatory cytokines, suchas TNF-.alpha., can induce DNA damage through reactive oxygen species(ROS) and nitrogen species, which leads to tumor initiation. TGF-.beta.is a powerful pleiotropic cytokine with immune suppressing andanti-inflammatory properties, inhibiting cell cycle progression andpromoting apoptosis. Inflammatory bowel diseases (IBD) are a major riskfactor for the development of colon cancer, by a mechanism called inliterature colitis-associated cancer (CAC). The increased prevalence ofCAC in IBD patients is influenced by disease severity and duration, andby the efficacy of anti-inflammatory therapies It seems that IBD areinduced and preserved by various microorganisms and frequently involvessigns of global dysbiosis, according to changes in the number, diversityand stability of microbiota. Increasing evidence shows that dysbiosisinduces the production of genotoxins and metabolites associated withtumorigenesis and produces disorder of the immune response whichpromotes and maintains inflammation in IBD leading to cancer.

Potassium diazoacetate, a stable form of nitrosated glycine, has beenfound to initiate mutations in the p53 gene, supporting the hypothesesthat NOC linked to glycine subscribes to p53 mutations in humans. Highlevels of polyamines are toxic and are associated with several diseases,including cancer, and oxidative stress that results from polyaminecatabolism is the underlying mechanism of toxicity. Several pathogens,including Shigella flexneri, Streptococcus pneumoniae, Salmonellaenterica and H. pylori, utilize polyamines to increase their virulence.One aspect of the present invention is to reduce the virulence ofvarious bacteria found in a person's microbiota by employing CRISPR-Cassystems (or similar systems) to modify, if not remove the virulenceabilities of various microbes to produce virulence factors.

Chronic inflammation can deeply alter local immune response and causethe liberation of nitric oxide. ROS can be produced by the gutmicrobiota or generated by immune cells during inflammation.Gastrointestinal bacteria generate nitric oxide from nitrate andnitrite. ROS are potent mutagens that lead to DNA breaks, pointmutations, and protein-DNA crosslinking and influence chromosomalinstability and the risk of cancer.

MicroRNA (miRNAs) are small (21-25 nucleotide) non-coding RNAs (ncRNAs)that regulate the translation and stability of their specific mRNAtargets. The aberrant expression of microRNAs is related to theinitiation and progression of various cancers, with MiRNAs acting astumor suppressors or oncogenes. Inflammation determines changes inexpression of miRNAs, primarily through the actions of proinflammatorycytokines. The role of miRNAs is believed to be in the initiation andprogression of human cancer, as well as in involvement with immuneresponses, inflammation, cell proliferation and cell death, all of whichare known to be regulated by NF.kappa.B. The overexpression of certainmiRNAs is believed to lead to the repression of tumor suppressor genesthat promote tumor survival and cell migration through NF-.kappa.Bactivation. While the gut microbiota interacts directly with the hostthrough the production of metabolites, peptides and other molecules, howmicrobiota regulates miRNA expression and contributes to the maintenanceof intestinal homeostasis and to IBD pathogenesis is still largelyunknown. miRNAs play a role in colonic carcinogenesis and theirreduction by butyrate is an important mechanism of its anti-cancereffects.

p53 is altered in many tumors. Various treatment strategies have focusedon targeting p53. About 50% of human tumors have TP53 gene mutations.Because formation of a tetrameric structure is critical forprotein-protein interactions, DNA binding, and the post-translationalmodification of p53, a small destabilization of the tetrameric structureresults in dysfunction of tumor suppressor activity.

p53 tumor suppression protein is sometimes called “the guardian of thegenome” as it is a key component of the cellular mechanisms controllingcellular responses to various cellular stresses. p53 is activated andprimarily functions as a transcriptional regulator of expression ofmultiple effector proteins and miRNAs, which, in turn, regulate keycellular processes such as apoptosis, cellular proliferation, andautophagy. Since regulation of cellular stress responses is tightlyintertwined with metabolic regulation, there is an interplay between p53and multiple pathways involved in regulation of metabolism and cellularhomeostasis that is complex and not fully understood. Approximately 50%of all human cancers have mutant p53, with approximately 75% of suchcancers having a single amino acid residue missense mutation in theDNA-binding core domain. The p53 protein recognizes and binds tospecific DNA response elements and activates gene transcription. The p53gene is the most frequently mutated gene in cancer and is atranscriptional factor that exerts broad anti-proliferative effects,including growth arrest, apoptosis, and cell senescence.

Certain embodiments of the present invention are directed to a methodthat restores p53 via its expression by an individual's microbiome, suchthat tumors can be treated. Such methods involve a step of exposingcancer cells to bacteria that produce p53 proteins. p53 tumor suppressorhas been identified as a protein interacting with the large T antigenproduced by simian vacuolating virus 40 (SV40) Inhibition of p53 can beachieved by bacterial pathogens which actively inhibit p53 protein andinduce its degradation, resulting in alteration of cellular stressresponses. This phenomenon was initially characterized in gastricepithelial cells infected with Helicobacter pylori, a bacterial pathogenthat commonly infects the human stomach and is strongly linked togastric cancer. Besides H. pylori, a number of other bacterial speciesinhibit p53.

Various embodiments of the present invention are directed towards thedirect interplay between bacterial pathogens and tumor suppressionmechanisms that protect an individual from cancer development. Variouspathogenic bacteria actively inhibit the major tumor suppression pathwaymediated by p53 protein that plays a key role in the regulation ofmultiple cellular stress responses and prevention of cancerogenesis.Bacterial degradation of p53 was first discovered in the context ofHelicobacter pylori infection, which is currently the strongest knownrisk factor for adenocarcinoma of the stomach. This phenomenon, however,is not limited to H. pylori, and many other bacterial pathogens inhibitp53 using various mechanisms. Inhibition of p53 by bacteria is linked tobacterial modulation of the host cellular responses to DNA damage,metabolic stress, and, potentially, other stressors.

Inactivation of p53 is a hallmark of tumorigenic changes. More than halfof all tumors carry p53 mutations, rendering the p53 gene (tp53) themost mutated gene in human tumors. p53 can also be inhibited bymutation-independent mechanisms Inhibition of wild-type p53 by the SV40virus was one of the first reported examples.

One aspect of the present invention is directed to bacterial inhibitionof p53. Recent studies have found that it is not only viruses, but alsosome pathogenic bacteria, that actively inhibit p53 and induce itsdegradation. This phenomenon was initially described in gastric cellsco-cultured with Helicobacter pylori. H. pylori is a gram-negative,spiral-shaped pathogen that lives in the stomachs of approximately halfof the world's population. The infection is typically acquired duringchildhood and causes lifelong chronic infection. Because of theassociation between H. pylori infection and the incidence of gastriccancer, the International Agency for Research on Cancer (IARC) hasclassified this bacterium as a Group 1 carcinogen. H. pylori infectionis considered to be the strongest known risk factor for gastric cancer,and epidemiological studies have estimated that, in the absence of H.pylori, 75% of gastric cancers would not occur.

H. pylori is able to dampen activity of p53 protein by inducing itsrapid degradation. One particular aspect of certain embodiments of thepresent invention is directed to modifying H. pylori, preferably via useof a CRISPR-Cas system, such that its abilities to degrade p53 arereduced. The supplantation of such modified H. pylori, preferably justafter a round of antibiotic treatment to reduce the numbers of nativeresident H. pylori in an individual's body, is done to then provide acompetitive advantage of such modified bacteria and thus, will result inthe reduction of p53 degradation, assisting in the treatment of thecancerous condition of the individual. Older people with gastricprecancerous lesions, who are infected with H. pylori, may beparticularly vulnerable to degradation of p53. H. pylori inhibits p53through multiple mechanisms, implying that inhibition of p53 activity isan important factor for successful infection. The bacteria not onlyinduce degradation of p53, but also alter the expression profile of p53isoforms. Other bacteria induce degradation of p53 using a similarmechanism to that of H. pylori. As such, the method as set forth hereincan be employed with other bacteria, as one of skill in the art willappreciate. For example, the intracellular bacterial pathogen Chlamydiatrachomatis as well as other Chlamydia species, induce degradation ofp53 by activating HDM2 protein. Degradation of p53 by Chlamydiacontributes to cancerogenesis in the female genital tract and inhibitionof p53 through the HDM2-dependent mechanism is employed byenteropathogen Shigella flexneri, which causes bacillary dysentery inhumans. Certain bacteria can inhibit transcription of the p53 gene. Suchinhibition of p53 may provide certain benefits to bacteria, such asallowing bacteria to subvert the host cell cycle control and apoptosismechanisms, resulting in inhibition of cell death and survival of hostcells damaged by infection. In the case of H. pylori, expression of theCagA virulence factor is sufficient to inhibit p53 and extend short andlong term survival of gastric epithelial cells that underwent DNAdamage. Thus, one embodiment of the preset invention is directed to themodification of H. pylori to remove or reduce the efficacy of the CagAvirulence factor such that an individual pretreated with antibiotics toreduce resident H. pylori, followed by administration of H. pylori tosuch an individual where modifications to such modified strain has beenmade (e.g. via CRISPR tools) results in a better treatment method forparticular types of cancer. The p53 pathway is known to affect immuneresponse. Among direct transcription targets of p53 are a number ofproteins regulating innate immunity and cytokine and chemokineproduction. p53 is also known to affect NF-.kappa.B activity andpro-inflammatory signaling.

One aspect of certain embodiments of the present invention is directedto the role of immunomodulatory function involved in the bacterialinhibition of p53. Some bacteria have evolved to inhibit p53 and do sovia multiple mechanisms, including protein degradation, transcriptionalinhibition, and post-translational modifications. p53 inhibition affectsthe host immune response, permitting bacteria to thrive and establishthemselves. p53 has a role in controlling the bacterial infections andthe inhibition of p53 confers certain selective advantages to bacteriabut causes an increase in the risk of tumor development, especially whenthere exist conditions of prolonged chronic infections.

Numerous bacterial pathogens have also been shown to inactivate themajor tumor suppressor p53 during infection. Such inactivation impedesthe protective response of the host cell and affects the downregulationof host cell metabolism to interfere with intracellular bacterialreplication, highlighting the crucial role of p53 in host-pathogeninteractions.

Yet other aspects of the present invention are directed to the linksbetween poor oral health and periodontal disease with an increased riskfor cancers. The present inventors believe that periodontal diseasecontributes to the development of systemic inflammation and if leftuntreated, a chronic, smoldering inflammatory response occurs inresponse to periodontal microbial pathogens and their products, such asendotoxin. Infection will ultimately stimulate the production ofpro-inflammatory cytokines and mediators such as IL-1.beta., IL-6,TNF-.alpha. and MMPs. IL-6, in particular, has tumor-inducing actions,by promoting growth and proliferation, in both healthy and malignantcells. IL-1 promotes tumor growth and metastasis by inducing matrixmetalloproteinase activity and other growth factors.

Intestinal bacteria are implicated in several types of cancer.Helicobacter species have been associated with enhanced carcinogenesisincluding liver cancer, colon cancer, and mammary carcinoma. Many humanviruses are known to impair p53 activity. In cervical cancer, the humanpapillomavirus E6 protein targets p53 for degradation. Bacterialinfection has been shown to trigger the p53 pathway and to activate p53isoforms. Resveratrol has been shown to inhibit carcinogenesis throughthe induction of p53-dependent cell death.

In one aspect of the present invention, using CRISPR-Cas, a more stablep53 protein is constructed in terms of the stability of its foldingbeing preserved, thus protecting its tumor suppressing function.According to the present invention, the regions where the p53 proteinare most vulnerable to mutations that cause improper folding aretherefore targeted and revised so as to impede common mis-foldingevents. Using such an improved, stable form of p53, and having itexpressed in an individual's microbiome, is an important aspect of thepresent invention as approximately 50% of all human cancers have mutantp53. Tumor initiation and maintenance depend upon inactivation of p53.Thus, certain embodiments of the present invention are directed to amethod that restores effective amounts of p53 to a person via anindividual's microbiome so that p53 can deter cancer cell proliferationand shrink tumor volume. One way in which to accomplish this objectiveis to have gut microbes produce amounts of p53 such that effectiveamounts thereof are available to deter cancers. Another route is toprovide a modified version of p53 that is more stable and thus, lesssusceptible to being degraded by bacteria.

More than half of all tumors carry p53 mutations, rendering the p53 gene(aka tp53) the most mutated gene in human tumors. p53 can also beinhibited by mutation-independent mechanisms. Viruses, as well as somepathogenic bacteria, actively inhibit p53 and induce its degradation.This phenomenon was initially described in gastric cells co-culturedwith Helicobacter pylori, a gram-negative, spiral-shaped pathogen thatlives in the stomachs of approximately half of the world's population.The infection is typically acquired during childhood and causes lifelongchronic infection. H. pylori infection is considered to be the strongestknown risk factor for gastric cancer, and epidemiological studies haveestimated that, in the absence of H. pylori, 75% of gastric cancerswould not occur. H. pylori is able to dampen activity of p53 protein byinducing its rapid degradation. One aspect of certain embodiments of thepresent invention is therefore directed to the provision of modified H.pylori bacteria to an individual such that such modified bacteria,lacking its native form ability to dampen the activity of p53, is usedto populate the microbiome (e.g. gut microbiome) of an individual sothat cancers associated with H. pylori are reduced. Moreover, H. pylorimodified via CRISPR-Cas to express p53 protein, is one method forensuring that cancer rates in individuals remain low. The population ofan individual's microbiome with such modified bacteria is one way inwhich to alter the conventional microbiome of the person in a mannerthat lessens the risk of cancer.

As it is known that older people with gastric precancerous lesions, whoare infected with H. pylori, may be particularly vulnerable todegradation of p53, the treatment of such individuals with modified H.pylori bacteria can alter the course of various diseases, includingcancer. H. pylori inhibits p53 through multiple mechanisms. In the caseof H. pylori, expression of the CagA virulence factor is sufficient toinhibit p53 and extend short and long term survival of gastricepithelial cells that have DNA damage Inhibition of p53 through theHDM2-dependent mechanism is employed by enteropathogen Shigellaflexneri, which causes bacillary dysentery in humans. Thus, treatment ofsuch disease states with modified bacteria able to produce desiredamounts of competent (e.g. effective, non-mutated, but more stable p53proteins) is one aspect of the present invention. Thus, in addition tocancer treatments, the modification of certain bacteria to address thelevels of p53 expressed thereby is an important aspect of variousembodiments of the present invention. For example, down-regulation ofp53 protein has been reported in studies of Neisseria gonorrhoeae, whichis responsible for the sexually transmitted gonorrhea that may increasethe risk of genital neoplasms. N. gonorrhoeae causes strong genotoxicstress and induces both single and double strand DNA breaks, which isbelieved to be associated with and can inhibit transcription of the p53gene.

The p53 pathway is known to affect immune response and among directtranscription targets of p53 are a number of proteins regulating innateimmunity and cytokine and chemokine production. p53 is also known toaffect NF-.kappa.B activity and pro-inflammatory signaling. One aspectof certain embodiments of the present invention therefore involve therole of immunomodulatory function involved in the bacterial inhibitionof p53 which affects the host immune response and permits bacteria tothrive.

In certain embodiments, the use of CRISPR-Cas or cpf1 system is employedto achieve targeted gene deletion for tailoring bacteria for cancertherapy. The above discussion with respect to modifying p53 proteinproduction, e.g. so as to render p53 proteins more stable and lesssusceptible to degradation, is an example of how CRISPR-Cas systems canbe employed to achieve this objective. A bacterial strain is preferablydesigned by the use of CRISPR-Cas systems in a way that themicroorganism is both attenuated and optimized at the same time. Forexample, auxotrophic/attenuated bacteria may express a complementinggene under an inducible promoter, such that their activation depends onpresence of, for example, arabinose or anhydrotetracycline, and thus,such bacteria can be inducibly complemented.

In some embodiments, Gram-negative bacteria, like Salmonella, areemployed as active delivery vehicles and preferably, instead ofdepending upon lysis to deliver the contents of the bacterium, acontrolled release of a therapeutic compound is facilitated in a mannerthat achieves continuous expression and release of a therapeuticcompound (e.g. such as tomatidine or p53) with a desired highconcentration over a period of time to effect cancer reduction, muscleatrophy treatment, etc. One objective is to deliver therapeuticcompounds actively and directly to the site of interest by usingbacteria of the individual's inherent microbiome. Thus, one aspect ofthe present invention is directed to the exploitation of the uniquetumor colonizing property of bacteria to achieve drug delivery viabacterial mediated tumor therapy. In certain preferred embodiments, useof bioluminescent bacteria are employed to follow the course of themicroorganisms into the tumor. While applicable for a number of cancers,in one embodiment, a CRISPR-Cas or cpf1 modified bacterium is used inthe treatment of pancreatic cancer with Listeria monocytogenes. Thus, incertain embodiments, bacteria are designed to deliver therapeuticcompounds like chemotherapeutic drugs directly into the canceroustissue. In various embodiments, bacteria that reside in an individual'smicrobiome (e.g oral, gut, vaginal, skin, etc.) are employed as vectorsystems that provide therapeutic compounds to cancer sites, includingsolid tumors and in a manner that is far more efficacious than, and thatovercomes, the limitations of conventional therapies.

Yet another example of cancer treatments employed using the presentinvention is the treatment of esophageal adenocarcinoma (EA), which hasincreased 6-fold in the U.S. since the 1970s, as well as pancreaticcancers. No one knows why. High antibody levels for one of the moreinfectious periodontal bacterium strains of Porphyromonas gingivalishave been associated with a two-fold risk for pancreatic cancer.Individuals with high levels of antibodies for some kinds of harmless“commensal” oral bacteria were associated with a 45-percent lower riskof pancreatic cancer. Thus, one aspect of the present invention isdirected to the reduction in pancreatic cancer via modification of anindividual's microbiome, and in particular their oral and gutmicrobiomes. Administration of modified oral and gut bacteria havingdesired characteristics as described herein is one way in which toreduce the incidence of pancreatic cancer.

Yet another aspect of the present invention is directed to the use ofhuman specific species of bacteria that are then modified to enhance oneor more characteristics deemed beneficial to the microbiome of anindividual, including bacteria that have been modified via a CRISPR-Cas9and/or Cpf1 systems (CRISPR-Cas12a) to either repress the expression ofa particular protein or lipid, or to increase the production ofbeneficial microbial secretions, including but not limited to tomatidineand p53 protein. One objective of such embodiments is to avoid modifyingan individual's human genome in order to treat a disease state. One canavoid modifying the human genome and still significantly affect thehealth of humans by instead employing modifications to the skin, oraland gut microbiomes. Use of human specific strains of bacteria, whetherthey are commensal or pathogenic, including bacteria that are modifiedto alter their native pathogenicity, is one preferred aspect of manyembodiments of the present invention. In particular, in view of thetropism demonstrated by S. pyogenes for humans, and the recognition thatsuch bacterial species is found in both the oral and skin microbiome ofhumans, S. pyogenes is a preferred bacterial species to employ invarious embodiments of the present invention to treat various diseasestates.

In various embodiments, re-cultivated human intestinal microbiotaobtained by cultivation of a stool sample in a cultivation medium isemployed to promote the proliferation of select bacteria, including atleast two of the following Phyla: Bacterioidetes, Firmicutes,Proteobacteria and Actinobacteria, and more preferrably at least two ofthe following: Faecalibacterium, Lachnospira, Veillonella, Rothia;LactoBacillus johnsonii and Prevotella. In other embodiments, one ormore of the following microorganisms is employed: Bifidobacteriumlognum, B. infantis BCRC 14602; Prevotella; Ruminococcus,Bifidobacterium infantis, LactoBacillus acidophilus, Bacteroidesfragilis, B. longum bv. infantis isolate UCD272; B. infantis BCRC; B.longum bv. infantis, AY151398; and LactoBacillus ruminus; L. lactis, L.lactis cremoris, L. plantaru, and L. raffinolactis; Faecalibacterium,Lachnospira, Veillonella, and Rothia; LactoBacillus johnsonii,LactoBacillus crispatus, LactoBacillus brevis, LactoBacillus buchneri,LactoBacillus casei, LactoBacillus delbrueckii, LactoBacillus fermentum,LactoBacillus helveticus, LactoBacillus kefir, LactoBacillus paracasei,LactoBacillus plantarum, LactoBacillus rhamnosus, LactoBacillussalivarius, Streptococcus thermophilus, Lactococcus lactis, Lactococcusplantarum, Lactococcus raffinolactis, Leuconostoc lactis, Leuconostocmesenteroides, Enterococcus faecalis, and Enterococcus faecium;Enterococcus faecalis; LactoBacillus reuteri, and LactoBacillusparacasei. In certain embodiments, the method includes the use of amixed culture of bacterial cells of three to eight species of lacticacid bacteria. In particular mixed cultures, the following may beincluded: Saccharomyces cerevisiae, LactoBacillus delbrueckii,LactoBacillus acidophilus, LactoBacillus plantarum, LactoBacillusfermentum, LactoBacillus casei, LactoBacillus rhamnosus, Lactococcuslactis and Streptococcus thermophilus; Enterococcus faecium; Bacilluscoagulans; Leuconostoc, Pediococcus, LactoBacillus casei, LactoBacillusplantarum, Lactococcus lactis subspecies lactis, Lactococcus lactissubspecies cremoris; LactoBacillus plantarum; Pediococcus pentosaceus;Streptococcus thermophilus; LactoBacillus paracasei; LactoBacillusplantarum, LactoBacillus gasseri and LactoBacillus salivarius;LactoBacillus acidophilus PM-A0002, LactoBacillus gasseri, LactoBacillussalivarius, LactoBacillus acidophilus PM-A0013; Leuconostocmesenteroides; LactoBacillus bulgaricus, LactoBacillus rhamnosus,LactoBacillus acidophilus, LactoBacillus paracasei; Bifidobacteriumbifidum; LactoBacillus brevis; Enterococcus durans, Leuconostocmesenteroides; LactoBacillus crispatus. Still other embodiments of theinvention may comprise extracts obtained from one or more of thefollowing species: LactoBacillus fermentum, LactoBacillus rhamnosus,LactoBacillus plantarum, LactoBacillus johnsonii, LactoBacillushelveticus, LactoBacillus casei defensis, LactoBacillus casei ssp.casei, LactoBacillus paracasei, LactoBacillus bulgaricus, LactoBacillusparacasei, LactoBacillus acidophilus, LactoBacillus reuteri,LactoBacillus salivarius, and LactoBacillus lactis. In some embodiments,at least one strain from each of the above species of bacteria is used,while in other embodiments, one or more specific strains from the listabove may be removed or substituted with one or more different strains.In particular, some embodiments of the present invention comprise anextract obtained from one or more of the following bacterial strains:LactoBacillus fermentum 1-3929, LactoBacillus rhamnosus 71.38,LactoBacillus plantarum 71.39, LactoBacillus johnsonii 103782, andLactoBacillus helveticus 103146; LactoBacillus fermentum 1-3929,LactoBacillus rhamnosus 71.38, LactoBacillus plantarum 71.39,LactoBacillus johnsonii 103782, and LactoBacillus helveticus 103146. Thefollowing bacteria species may also be employed: LactoBacillusacidophilus PM-A0002 deposit number M 207038, LactoBacillus gasseriPM-A0005 deposit number M 207039, LactoBacillus salivarius PM-A0006deposit number M 207040, LactoBacillus johnsonii PM-A0009 deposit numberM 207041 and LactoBacillus acidophilus PM-A0013 deposit number M207042.Certain other embodiments of the present invention include a combinationof particular bacterial strains, selected from the group consisting ofPrevotella; LactoBacillus johnsonii; Bacteroides fragilis, LactoBacillusruminus, and at least one of B. longum bv. infantis isolate UCD272 or B.longum bv. infantis, AY151398. In more preferred embodiments, the gutmicrobiome of an individual is modified by providing in preferably apill form a collection of microbes that include at least two of thefollowing Phyla: Bacterioidetes, Firmicutes, Proteobacteria andActinobacteria, and more preferrably at least two of the following:Faecalibacterium, Lachnospira, Veillonella, Rothia; LactoBacillusjohnsonii and Prevotella.

As one of ordinary skill in the art will appreciate, one must give valueto their existence by behaving as if one's very existence were a work ofart. You must have chaos within you to give birth to a dancing star. Andthose who were seen dancing were thought to be insane by those who couldnot hear the music. To live is to suffer, to survive is to find somemeaning in the suffering. No one can construct for you the bridge uponwhich precisely you must cross the stream of life, no one but youyourself alone. There will always be rocks in the road ahead of us. Theywill be stumbling blocks or stepping stones; it all depends on how youuse them. The center is everywhere. Bent is the path of eternity.

In still other embodiments, interspecies interactions within mixedmicrobial communities is involved, with the objective being to modifycompetitive relationships involving nonbiocidal biosurfactants, enzymes,and metabolites produced by bacteria and other microorganisms in amanner such that selection of particular bacterial species can beemployed to do one or more of inhibit initial adhesion, trigger matrixdegradation, encourage jamming of cell-cell communications, and inducebiofilm dispersion. Nonbiocidal molecules are thus employed to modifycompetitive interactions within biofilms in a manner that promotes theoverall health of an individual's microbiome.

In certain embodiments, particularly designed to address the properdevelopmental biology of a human's immune system, so important in earlyand later life ability to thwart cancerous conditions, a bacterialformulation is applied to newborns within a critical window of timeafter birth, preferably within the first 24 hours of the newborn'sbirth, more preferably within 6 hours of their birth, even morepreferably within 3 hours of birth, and most preferably within an hourafter their birth. The administration can be by several methods, butpreferably is a lotion, ointment or gel that is rubbed onto thenewborn's skin, preferably all over his/her entire body. A spray or mistcan also be applied that contains the bacterial and microbe formulationsas set forth herein. While not bound by theory, the critical window toapply to the newborn's skin the referenced formulations, e.g. microbialmixtures of bacteria beneficial in triggering immune system developmentas further described herein, is within a relatively short time periodand is necessary to establish immune tolerance to a variety of commensalmicrobes. The manner in such and the content of microbes presented at atime in which a newborn has his/her skin, oral cavity and gut microbiomecolonized establishes immune tolerance to particular commensal microbes.The influx of highly activated T cells into neonatal skin and gut isbelieved to occur in such critical window. So a mother of a newborn hasa choice: to simply rely upon chance as to what particular microbesmight be present during this critical window of the newborn'sestablishing immune tolerance to particular bacteria and other microbes;or to provide the newborn with a selected formulation containingpredetermined microbes such that the newborn's developing immune systemcan properly react to the microbes in the predetermined formulation, andthus provide the newborn with the opportunity to develop a moreexpansive immune tolerance profile. The mechanism that promotestolerance is tissue specific, and thus, the skin, oral cavity and thegut may have different ways by which to mediate tolerance to commensalmicrobes. For example, to establish a healthy status of a newborn's skinas it relates to commensal microbes on its skin, the particular type ofmicrobes, including bacteria, brought into contact with his/her skin isachieved in a certain time period after birth (e.g. within 1 to 24 hoursafter birth) so that the developing immune system of the infantestablishes tolerance to such microbes, thus avoiding allergies,autoimmune diseases and other related diseases, as well as chronicinflammation of the skin.

In certain embodiments of the present invention, the skin/oral and gutmicrobiome is enhanced via providing microbes able to metabolize lipids,proteins and carbohydrates, and thus, with respect to the skinmicrobiome, produce acid that aids in maintaining the so-called “acidmantel” of the skin. In preferred embodiments the bacteria that ismodified has a very narrow host tropism, such that the bacteria arespecific for the human species and thus, their modification poses littleif any risk to other animals or organisms.

Other embodiments are directed to combating infections of a person'sskin by the bacteria Staphylococcus aureus. Patients with malignanciesrepresent a population at high risk for drug-resistant infections. S.aureus is a significant cause of morbidity and mortality in pediatriconcology patients. Staphylococcus aureus is a commensal and pathogen ofboth humans and cattle. In certain embodiments the accessory generegulator (Agr) system and the virulence regulation of S. aureuspathogenesis is modified to delete or to at least reduce the virulenceof the bacteria. In such a way, the present invention provides a way toeffectively combat S. aureus infections. In various embodiments of thepresent invention, CRISPR-Cas9 and/or Cpf1 systems are employed torender ineffective the virulence factors of such bacteria involved withthe establishment and propagation of infection. Several molecules havebeen found to interfere with S. aureus virulence regulation, especiallythose targeting the Agr quorum-sensing signaling molecule. Bymodification of this bacterial species using CRISPR-Cas and/or Cpf1 itis possible to achieve broad-spectrum inhibitory effects on most S.aureus strains and Agr subtypes.

The tropism of individual bacteria for particular host tissues (e.g.,skin vs. respiratory tract vs. gastrointestinal tract) is determined bythe array of available adhesion-receptor pairs. In preferredembodiments, bacteria having substantial, if not entire, human hostspecificity are employed. For example, Salmonella enterica serovarTyphi, known to be the bacteria responsible for typhoid fever, alife-threatening human disease, demonstrates strict human hostspecificity. In certain embodiments, the virulence factors of suchbacteria are compromised by being modified via the CRISPR-Cas or Cpf1system to render the modified bacteria as non-pathogenic. Similarly, thebacteria Neisseria, the causative agent of gonorrhea, is a diseaserestricted to humans, and thus similar CRISPR-Cas and/or Cpf1 systemsmay be employed to reduce if not eliminate the virulence factors of suchbacteria. Likewise, Helicobacter pylori is known to be an etiologicagent of gastritis and peptic ulcer disease in humans. The ironacquisition system of H. pylori by the human lactoferrin receptor systemis believed to play a major role in the virulence of H. pyloriinfection. The CRISPR-Cas and/or Cpf1 systems may be employed to reduceif not eliminate the virulence factors of this bacteria. Yet anotherbacteria demonstrating human tropism is Haemophilus influenza, a Gramnegative species that requires heme and has exclusive human hostspecificity. The precise way in which to employ CRISPR-Cas systems isrelatively straightforward and is described in great detail in variousreferences that are incorporated herein for written description andenablement purposes. In certain embodiments, the CRISPR-Cas and/or Cpf1systems may be employed to reduce if not eliminate the virulence factorsof such bacteria. The distinction between throat and skin group AStreptococcus has become blurred and to date there have been fewadvances in treatment of group A Streptococcus skin infections. Certainaspects of the present invention include the modification of skin groupA Streptococcus to reduce the likelihood, if not prevent, related skindiseases, including eczema, atopic dermatitis, acne, allergicinflammation, skin hypersensitivity, UV-induced skin damage, skincancer.

The present invention in various embodiments is directed to a variety ofconsumer products including cosmetic products such as skin care products(bath preparations, skin washing and cleaning products, skin careproducts, eye cosmetics, lip care products, nail care products, intimatehygiene preparations, foot care), those with special effects(sunscreens, tanning agents, deodorants, anticholinergics, depilatories,shaving, fragrance), those for oral or dental hygiene and those for haircare (shampoos, conditioners, etc. One objective of the presentinvention is to achieve various health and cosmetic benefits byproviding a healthy, balanced skin, oral and gut microbiome. Otherembodiments are directed to prebiotic agents for use with thosemicrobiomes. In preferred embodiments, CRISPR-Cas and/or Cpf1 modifiedbacteria, especially those demonstrating total or substantial tropismfor humans, are employed in one or more of the above referencedproducts, with certain features, namely, virulence factors, reduced ifnot eliminated. In such a manner, there is a competitive inhibition ofundesired bacteria with the modified bacteria as set forth herein.

In certain embodiments, the cleansing of one's skin to effectivelyreduce by at least about 50%, more preferably about 30%, and mostpreferably to reduce by at least about 25%, of native bacteria on anindividual's skin portion to be addressed, is performed prior topurposefully contacting the individual's skin with one or more bacteriaspecies that have been modified via employment of a CRISPR-Cas and/orCpf1 system to reduce if not effectively compromise the virulencefactors of such bacteria, and more preferably a bacteria that has a hostspecificity exclusive to humans. Similarly, treatment of the oral andgut microbiome can be addressed by such an initial reduction in thenative bacterial and other microbe populations of an individual,followed by repopulation of such microbiomes with desired modifiedbacteria, especially those modified via CRIPSR-Cas systems.

The adherence to the skin of problem flora, such as pathogenic bacteriaand yeast, has been associated with numerous ailments, including skininfections, diaper rash, urinary or vaginal infections, and malodors.Various products are commercially available to clean the surface of skinand to remove problem flora therefrom. Many presently available productsinclude an antibacterial agent, such as an organic acid, which can beused in combination with the surfactant to kill bacteria located on theskin's surface. Also, various antibacterial soaps and cleansers areavailable to cleanse hands and kill flora adhered to the skin's surface.These antibacterial soaps are generally highly effective in killingbacteria located on the skin.

Various embodiments of the present invention stand in contrast toaccepted methods of dealing with skin and bacteria issues (which largelysolely involve killing bacteria, etc.—such as described in KimberlyClark's U.S. Pat. No. 8,110,215 to Koenig, et al.) In contrast, variousembodiments of the present invention are directed to modification ofvarious bacteria on a person's skin (and in still other embodiments tothe gut and oral microbiome) so as to reduce the pathogenicity thereofand to rely upon competitive inhibition of such modified bacteria on theskin to further reduce the presence of pathogenic bacteria on anindividual's skin.

As for lotions of the present invention, in preferred embodiments, thereis an objective to limit if not preclude the use of phthalates, whichare extremely toxic and are believed to also be human carcinogens. Thus,in preferred embodiments of the present invention, such lotions do notemploy such toxic agents, and in particular, agents toxic to bacterialspecies for which the inventors suggest be used, e.g. those modified toreduce pathogenicity, virulence factors, etc., so as to establish apopulation of such modified bacteria on a person's skin, and in such amanner, reduce the incidence of skin infections and diseases. Thus,lotions, creams, gels, etc. that include such toxic agents, includingbut not limited to phthalates, are not employed, but rather, lotionsthat provide an environment for the bacteria as set forth herein tosurvive and to thus be available to provide benefits to the skin ofindividuals to which they are applied, are particularly preferred.

Healthy, normal skin exhibits a slightly acidic pH in the range of4.2-5.6, which aids in the prevention of pathogenic bacterialcolonization, regulation of enzyme activity, and maintenance of amoisture-rich environment; however, after the age of 70, the pH of skinrises significantly, stimulating protease activity. Thus, one objectiveof several embodiments of the present invention is directed to loweringthe pH of the skin of an individual, especially those at about the ageof 70, so as to encourage a skin environment conducive to theproliferation of one or more bacteria that have been modified to promoteskin health and to reduce the ability of undesired bacteria fromcolonizing the skin of the person. Probiotic metabolism frequentlyproduces acidic molecules, lowering the pH of the surroundingenvironments seen with Lactobacilli producing free fatty acids (FFAs)and conjugated linoleic acid (CLA) during the fermentation process.Thus, the use of probiotics is employed to restore the normal skin pHand consequently return protease activity levels closer to those seen inyoung, healthy skin.

The main microbes that reside on human skin can be divided into fourphyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes.Staphylococcus spp. and Corynebacterium spp. are the dominant bacteriaat the genus level. Significantly fewer Corynebacterium spp. have beenobserved in cachexia patients compared to healthy subjects. The presenceof cancer and cachexia alters human skin bacterial communities.Understanding the changes in microbiota during cancer cachexia has leadto new insights into the syndrome. Especially with tomatidineadministration and enhanced bacteria, the provision of such modifiedbacteria to a person's microbiomes, including the gut, oral and skinmicrobiomes, provides a way to address numerous issues arising fromcancer cachexia.

Competitive inhibition is relied upon in various embodiments of thepresent invention to advance the repopulation of skin, oral cavity andgut environments with beneficial microbes. For example, and using theskin microbiome as one specific case, in one embodiment, repopulating anindividual's skin with beneficial bacteria, preferably in balancedpercentages and having preferred species provided, can be used inconjunction with an antimicrobial composition. Preferably, anantimicrobial is first administered to suppress or eradicate theresident populations of bacteria on a person's skin, including anyabnormal organisms or pathogenic bacteria, then the normal flora isrepopulated by the administration of at least one of the modifiedbacteria as described herein, including those modified using CRISPR-Casand/or Cpf1 systems to delete certain portions of genes or to addcertain genes to facilitate the colonization of a person's skin withbeneficial bacteria that maintain the general health of a person's skin.

The term “therapeutically effective amount” as used herein means theamount contained in the composition administered that is of sufficientquantity to achieve the intended purpose, such as, in the case ofcachexia, an amount that is able to reduce muscle wasting activitiesthat cause a loss of muscle weight in an individual.

It is preferred in many embodiments that antimicrobial treatments arecompleted before the administration of modified bacteria—selected asbeing desirable to maintain skin, oral or gut micriobiome health,including but not limited to modified bacteria of the following:Firmicutes (mainly Streptococcus and Staphylococcus) and Actinobacteria(mainly Corynebacterium and Propionibacterium). By employing suchmodified bacteria, one is able to establish and maintain the reductionif not preclusion of various skin diseases, including skin cancer. Oneobjective of certain aspects of the present invention is to provide amethod and system that, by using health promoting strains from themicrobiome in topical probiotics, it is possible to treat and to furtherreduce the risk of skin cancer. One of skill in the art will appreciatesimilar objectives in the treatment of the oral and gut microbiomes fordiseases that affect the same.

Repair of tissue wounds is a fundamental process to re-establish tissueintegrity and regular function. Infection is a major factor that hinderswound healing. Multicellular organisms have evolved an arsenal ofhost-defense molecules, including antimicrobial peptides (AMPs), aimedat controlling microbial proliferation and at modulating the host'simmune response to a variety of biological or physical insults. Certainembodiments of the present invention are directed to the use of AMPs asendogenous mediators of wound healing. Thus, one aspect of severalembodiments of the present invention is directed to geneticallymanipulating bacterial species native to the skin. Staphylococcusepidermidis, which is found in abundance on human skin, can cause immunetolerance in some—but in others, inflammation and activation of T cellsagainst the bacteria. The present inventors submit that the immunesystem may set up tolerance to commensal bacteria only early in life,during a time where there is an influx of regulatory T cells unique tothe skin, e.g. during the first week after birth. This colonization ofthe skin by regulatory T cells—immune cells that dampen the responses ofeffector T cells—is believed to be required for tolerance to S.epidermidis. There is an abrupt wave of regulatory T cell infiltrationinto neonatal skin that occurs at a defined period and this windowdictates the achievement of commensal-specific tolerance.

One aspect of the present invention is directed to the introduction oftolerance to commensal bacteria during the time the developmental windowis still open, thus providing the individual with life-long protectionfrom a variety of diseases. Still other embodiments, however, aredirected to introducing tolerance following the closing of thedevelopmental window, e.g. after the first week after birth, so thatindividuals can purposefully be induced to have commensal-specifictolerance as an adult. Understanding which microbes cause infection andwhich are tolerated and the critical time frames where the immune statusis set is one aspect of the present invention.

Skin bacterial communities are influenced by ethnicity, lifestyle and/orgeographic location. Skin bacterial communities that are particularlyemployed in the modifications as set forth herein include: Firmicutes,Proteobacteria and Actinobacteria); Firmicutes (mainly Streptococcus andStaphylococcus) and Actinobacteria (mainly Corynebacterium andPropionibacterium), while still other preferred bacteria include L.acidophilus NCFM, L. salivarius Ls-33, Bifidobacterium lactis 420, L.acidophilus La-14 and Propionibacterium jensenii P 63.

In various embodiments, cosmetics are provided that provide for a mediumfavorable for maintaining a desired physio-chemical balance of the skinwithout favoring the development of pathogenic microorganisms. Toachieve this objective, certain oligosaccharides that are metabolized byseveral beneficial strains of the skin microflora, such as Micrococcuskristinae, Micrococcus sedentarius, Staphylococcus capitis,Corynebacterium xerosis and LactoBacillus pentosus, are employed informulations, in conjunction with one or more of the modified bacteriaas described herein, including those modified to produce tomatidineand/or p53 proteins.

Pathogenic strains such as Staphylococcus aureus, Gardnerella vaginalisand Propionibacterium acnes do not typically metabolize, or veryslightly metabolize, certain oligosaccharides. In certain embodiments,sugar sources are provided in amounts and in association with beneficialbacteria, whether they be those modified as described herein, or thosethat are naturally non-pathogenic in nature, so as to achieve thecolonization of the skin in a fashion to provide the health benefitssought.

Yet another aspect of the present invention is directed to the treatmentof brain cancer, which is the leading cause of cancer-related death inpatients younger than age 35 and accounts for roughly 10% of all cancersdiagnosed in North America. Treatment of brain tumors is complicated bythe fact that there are more than 120 different types, which range fromlow grade astrocytomas to high grade glioblastomas (GBM). Malignantgliomas, such as GBM, are by far the most common brain cancer found inadults and one of the most difficult to treat. Even with aggressivesingle and multimodal treatment options such as surgery, chemotherapy,radiation and small molecule inhibitors, the survival has remainedunchanged over the past three decades with a median survival of lessthan one year after diagnosis. Reasons for the failure of conventionaltreatments is multifactorial including the highly infiltrative/invasivenature of GBM, limitation of drug delivery through the blood brainbarrier and neural parenchyma, and genetic heterogeneity resulting inintrinsic resistance to available treatments and the rise of aggressiveresistant clones.

To address such brain tumors, one aspect of the present invention isdirected to the delivery of tomatidine and/or p53 directly to tumorsthrough interstitial therapy, where a surgeon implants small e.g.dime-sized strips having the agent as desired, e.g. tomatidine, p53,etc. and the preferably biodegradable strips that comprise such agentsare delivered directly into the tumor so that they may release desiredconcentrations of the agent(s) locally over a period of days or weeks,prior to safely dissolving. Such strips can be customized to treat avariety of solid tumor disease of the breast, lung, colon, kidney andskin. The inventors incorporate by reference various novel technologiesrelating to the use of strips, such as oral strips as described in U.S.Pat. No. 9,010,340 and Ser. No. 14/611,458. One of skill in the art willappreciate the modifications to such strips to employ their use in thevarious cancer treatment regimens as described herein.

In one particular aspect of the present invention, the Zika virus isemployed in the treatment of brain cancer due to its ability to targethuman brain cells. Employing such targeting in combination with theother aspects of the present invention as described herein, includingthe production of tomatidine and/or p53 by an individual's microbiomes,offers new hope for an effective treatment of particular brain cancers.Zika virus is a member of the Flaviviridae family, which includes WestNile Virus, St. Louis encephalitis virus, Kunjin virus, yellow fevervirus, Dengue virus, and Japanese encephalitis virus. Cellular apoptosis(cell death) and necrosis follow infection for many of these viruses,and appears to be dependent upon several factors, such as viral load,host factors, and specific viral protein induced apoptosis/necrosispathways, many of which have yet to be fully defined. The expression ofBAX is regulated by the tumor suppressor p53. The majority of BAX isfound in the cytosol, but upon initiation of apoptotic signaling, BAXundergoes a conformational shift and becomes mitochondrial membraneassociated.

Still another aspect of the present invention is directed to the use ofparticular mushroom extracts to combat cancer, especially when combinedwith tomatidine to address the muscle atrophy commonly associated withcancer. Use of particular compounds derived from mushrooms, especiallythose produced by modified bacteria resident in an individual'smicrobiome and that are provided via the use of CRISPR-Cas or Cpf1systems, is a new way to address treatment of many cancer types. Forexample, the small-molecule neoalbaconol (NA) from Albatrellus confluenspossesses the ability to inhibit cell growth of many cancer cells.Neoalbaconol is a natural compound extracted from the mushroomAlbatrellus confluens, and induces necroptosis. Neoalbaconal inhibitsproliferation in various tumor cell lines, especially in breast cancerand nasopharyngeal carcinoma. Neoalbaconal targets PDK1 to inhibit thedownstream PI3-K/Akt pathway and blocks the generation of ATP in atime-dependent manner. Cholangiocarcinoma (CCA) is a lethal malignancywith poor prognosis that makes up 10-25% of all primary liver cancerdiagnosed worldwide. Albatrellus confluens, mainly distributed inSouthwest China, is a member of the Polyporaceae family. Severalcompounds with anticancer potential have been isolated from this fungusand NA has proven to be efficacious in inhibiting the growth of a broadspectrum of tumor cell lines. Dosage administration for mice would be NAtreatment (100 mg/kg/day)—and thus, for humans, would be commensuratewith the person's size being treated. When combined with tomatidine, aneffective treatment for cancer and one that addresses muscle atrophyassociated with cancer. Preferably, at least about 5 mg of tomatidineevery day is provided to an individual via the production thereof by gutmicrobes in such individual. Systemic administration of one or moredisclosed compounds (e.g., by parenteral injection or by oralconsumption) can be used to reduce fat, increase the muscle to fatratio, increase the muscle mass and reduce the fat, and prevent anincrease in fat in an animal.

It is known that most human viruses impair p53 activity. For example, incervical cancer, the human papillomavirus E6 protein targets p53 fordegradation. Bacterial infection triggers the p53 pathway and toactivate p53 isoforms and the p53 R249S variant is often observed inliver cancer as being associated with aflatoxin B1 food contamination.

In one aspect of the present invention, CRISPR tools are employed toinsert into cancer cells particular sequences that encode for theexpression of toxins. Appropriate promoters are used to then “turn on”the expression of such genes, thus enabling amounts of toxins to be madeby the cancer cell, destroying itself. In other words, this is similarin concept to “infecting” a cancer cell with a particular DNA or RNAinsert, whether in the nuclear DNA or in the mitochondrial DNA (or RNAof the cell) and by activating promoters to effectively “turn on” theproduction of the protein (e.g. a toxin), one can control thedestruction of the cell. Similarly, stretches of DNA can be insertedinto cancer cells such that when a later infection with a predeterminedbacterial species occurs, the normal immune response of the individual'sresident immune system will target such DNA stretches, and the targetingstep itself can be used to provide the cancer cells with suitablecomponents that ultimately reduce the growth of cancerous tissue and/orkill such cells. Other systems employ CRISPR tools to ensure that cancercells, when attempting to counter the infection by a virus, results inturning on destructive machinery that selectively kills the cancer cellin the process.

Yet other embodiments that are directed to the treatment of throatcancers employ factors of the disease causing bacteria Streptococcuspyogenes. For example, in one embodiment, the hyaluronic acid capsule ofStreptococcus pyogenes, along with its M proteins, which are a majorfactor behind its virulence due to their role in the attachment to hosttissues. Host immunity to Streptococcus pyogenes results from thedevelopment of antibodies specific to M protein and the hyaluronic acidcapsule of Streptococcus pyogenes is chemically similar to humanconnective tissue, which allows it to go unrecognized as an antigen bythe host's body. Adhesion of Streptococcus pyogenes to the host cell isthe first step in pathogenesis, and the invasion process into the hostcells takes place in very short order.

S. pyogenes adhesion to human cells depends on the presence of cellsurface adhesions including the M protein. The cell wall associated Mprotein is a major virulence factor of S. pyogenes, which can binddirectly to the extracellular matrix components (e.g. fibrinogen).

S. pyogenes possesses an arsenal of countermeasures against attacks fromthe host, including resistance of phagocytosis that is mediated by thehyaluronic acid capsule.

In various embodiments of the invention as described herein, componentsof CRISPR-Cas or Cpf1 systems are involved in the regulation ofbacterial gene expression. As Cas proteins have proven to be greatbiotechnology tools, these novel functions are used in variousembodiments of the present invention for gene regulation of bacteriathat comprise the human microbiome. In particular, a particular class ofriboswitches, called thermosensors can sense temperature changes and canbe used effectively with especially gram-positive bacteria, in contrastto Gram negative bacteria, which use translational attenuation.

Still another aspect of the present invention is directed to theproduction of rapamycin, a small molecule drug derived from Streptomyceshygroscopicus, by bacteria in a person's microbiome. Much of the roleand function of mTOR has been ascertained with rapamycin, a knownmacrolide antibiotic produced by Streptomyces hygroscopicus. Themechanistic target of rapamycin (mTOR) is an evolutionarily conservedserine/threonine kinase that is ubiquitously expressed in immune cells.mTOR integrates multiple environmental signals to regulate diversecellular processes including protein translation, cell growth,proliferation, metabolism, migration, and survival. Bacterial pathogensincluding Listeria monocytogenes (L. monocytogenes) and Staphylococcusaureas can also activate mTOR to promote IL-10 production and increasetheir survival in the host.

Isolated from Streptomyces hygroscopicus var. Ascomycetes, pimecrolimusis a calcineurin inhibitor which inhibits T-cell stimulation byantigen-presenting cells, blocking both T helper cell 1 (Th1) cytokinessuch as IL-2 and interferon (IFN-.gamma.) and T helper cell 2 (Th2)cytokines including IL-4 10. It also inhibits mast cell release ofhexosaminidase, tryptase, and histamine. Topical pimecrolimus, liketopical glucocorticoids, improves the atopic dermatitis-like skinlesions and barrier impairment (important for asthma development inatopic dermatitis patients), by suppressing thymic stromallymphopoietin-(TSLP-) related allergic inflammation. TSLP is anepithelial cell-derived IL-7-like cytokine which has an important rolein allergic inflammatory immune response, particularly in dendriticcell-mediated allergic inflammation in allergic asthma and atopicdermatitis, since it converts human epidermal Langerhans cells intoantigen-presenting cells which than induce proallergic T-cells.

Pimecrolimus is used in the treatment of Atopic dermatitis (the only oneindication approved by the FDA), but it has been effectively used totreat erosive oral and genital lichen planus, vulvar lichen sclerosus,Fox-Fordyce disease, intertriginous psoriasis, seborrheic dermatitis,erosive circinate balanitis, discoid lupus erythematosus, vulvarpruritus, vitiligo, and GVHD. Pimecrolimus has similar efficacy as lowto moderately potent topical corticosteroid creams for mild to moderateAtopoic dermatitis during the first 5 to 6 years of life, with similarrates of adverse events.

Topical calcineurin inhibitors are primarily used in the treatment ofAtopic dermatitis, which is not defined as a genetically predisposed dryhypersensitive skin any more, but as acute eczematous skin, whichemphasizes the crucial role in skin barrier integrity and extrinsicAtopic dermatitis. Topical therapy includes basic therapy that enhancesthe restoration and maintenance of the epidermal barrier.

In certain embodiments of the present invention, antibiotic resistanceof certain bacteria is modulated by employment of CRISPR to insert intothe genome of a bacteria antibacterial sensitivity such that it canselectively be killed, if necessary, after it is employed to triggerdesired immune responses in a new born or other individual.

CRISPR-Cas systems employ CRISPR RNAs to recognize and destroycomplementary nucleic acids. In various embodiments of the presentinvention, CRISPR-Cas systems are used as programmable antimicrobials toselectively kill bacterial species and strains such that desiredselected targets can be focused on such that virtually any genomiclocation may be a distinct target for CRISPR-based antimicrobials, andthat, in conjunction with an appropriate delivery vehicle, such as thoseemployed by Bikard et al. and Citorik et al., one is able to effectivelydeploy a CRISPR-Cas system as an antimicrobial agent.

Another aspect of certain embodiments include making syntheticCRISPR-containing RNAs that target genes of interest and using them withCas enzymes. The specificity of CRISPR-Cas systems permits one to designmethods to target a single bacterial species so that only essentialgenes from that one species is targeted and cut up. CRISPR-Cas systemsare employed in various ways in the many embodiments of the presentinvention to retain the beneficial bacterial communities intact and tooffer protection against undesired bacterial pathogens.

CRISPR has a certain protein in it called Cas9 that acts like a scissoras it recognizes specific sequences of DNA and cuts it enabling one toperform genome-editing of a bacterial genome in a person's microbiome.There exists another CRISPR system, CRISPR-Cpf1 that is even morepreferred for use in microbial systems. Cpf1 is important in bacterialimmunity and is well adapted to slice target DNAs. Cpf1 prefers a “TTN”PAM motif that is located 5′ to its protospacer target—not 3′, as perCas9, making it distinct in having a PAM that is not G-rich and is onthe opposite side of the protospacer. Cpf1 binds a crRNA that carriesthe protospacer sequence for base-pairing the target. Unlike Cas9, Cpf1does not require a separate tracrRNA and is devoid of a tracrRNA gene atthe Cpf1-CRISPR locus, which means that Cpf1 merely requires a cRNA thatis about 43 bases long—of which 24 nt is protospacer and 19 nt is theconstitutive direct repeat sequence. In contrast, the single RNA thatCas9 needs is still .about.100 nt long. Cpf1 is apparently directlyresponsible for cleaving the 43-base cRNAs apart from the primarytranscript.

With respect to the cleavage sites on the target DNA, the cut sites arestaggered by about 5 bases, thus creating “sticky overhangs” tofacilitate gene editing via NHEJ-mediated-ligation of DNA fragments withmatching ends. The cut sites are in the 3′ end of the protospacer,distal to the 5′ end where the PAM is. The cut positions usually followthe 18th base on the protospacer strand and the 23rd base on thecomplementary strand (the one that pairs to the crRNA). In Cpf1 there isa “seed” region close to the PAM in which single base substitutionscompletely prevent cleavage activity. Unlike the Cas9 CRISPR target, thecleavage sites and the seed region do not overlap. One advantage of thepresent invention, as compared to techniques that rely on CRISPR systemsto modify mammalian cells, is that the system and method of preferredembodiments are directed to bacterial systems—rather than eukaryoticsystems. It is believed that Cpf1 may be better than Cas9 for mediatinginsertions of DNA, namely because its guide RNA is only 43 bases long,making it feasible to purchase directly synthesized guide RNAs for Cpf1,with or without chemical modifications to enhance stability.

The CRISPR system may be employed in various embodiments to strengthenantibiotics or to kill the bacteria altogether. By removing thebacteria's genes that make them antibiotic-resistant, CRISPR can boostthe effectiveness of existing drugs. CRISPR can also be used to remove abacteria's genes that make them deadly and facilitate RNA-guidedsite-specific DNA cleavage. Analogous to the search function in modemword processors, Cas9 can be guided to specific locations within complexgenomes by a short RNA search string.

In various embodiments, the CRISPR-Cas systems is employed to controlthe composition of the gut flora, such as by circumventing commonlytransmitted modes of antibiotic resistance and distinguishing betweenbeneficial and pathogenic bacteria. For applications that require theremoval of more than one strain, multiple spacers that target shared orunique sequences may be encoded in a single CRISPR array and/or sucharrays may be combined with a complete set of cas genes to instigateremoval of strains lacking functional CRISPR-Cas systems. Because of thesequence specificity of targeting, CRISPR-Cas systems may be used todistinguish strains separated by only a few base pairs. Thus, in manyembodiments, CRISPR-Cas systems provide for the selective removal ofmicroorganisms to trigger certain predictable development of the immunesystem.

The specificity of targeting with CRISPR RNAs may be employed to readilydistinguish between highly similar strains in pure or mixed cultures.Thus, in certain embodiments, varying the collection of delivered CRISPRRNAs is employed to quantitatively control the relative number ofindividual strains within a mixed culture in a manner to circumventmultidrug resistance and to differentiate between pathogenic andbeneficial microorganisms.

Use of CRISPR-Cas provides a generalized and programmable strategy thatcan distinguish between closely related microorganisms and allows forfine control over the composition of a microbial population for use inthe present invention. Thus, the RNA directed immune systems in bacteriaand archaea called CRISPR-Cas systems is employed in various embodimentsof the present invention to selectively and quantitatively removeindividual bacterial strains based on sequence information. Thus, suchgenome targeting using CRISPR-Cas systems allows one to specificallyremove individual microbial species and strains.

In various embodiments, it is desirable to remove—using CRISPR-Cassystems—particular pathogenic bacteria and/or simply the pathogenicportions of such bacteria—while sparing other desired commensalbacteria.

In various embodiments, one of skill in the art will appreciate thatremoval of particular strains of bacteria may be achieved using bothtype I and type II CRISPR-Cas systems, given the distinction betweenthese systems being that type I systems cleave and degrade DNA throughthe action of a 3′-to-5′ exonuclease, whereas type II systems onlycleave DNA. In still other embodiments, multiple guide RNAs can also beused to target several genes at once. The use of effector fusions mayalso expand the variety of genome engineering modalities achievableusing Cas9. For example, a variety of proteins or RNAs may be tetheredto Cas9 or sgRNA to alter transcription states of specific genomic loci,monitor chromatin states, or even rearrange the three-dimensionalorganization of the genome.

CRISPR-Cas can be used on the various identified microbiome constituentsto modify gene expression, including cutting of a gene, repress oractivate a gene, etc. It can be employed to deliver desired regulatorsor any protein to a desired place on a genome of a microbe, thuspermitting one to tailor the attributes of the microbiome of anindividual to promote the health thereof. Because CRISPR-Cas acts beforetranscription occurs, it is able to be employed to target regulatory andother elements on the DNA of microbes that make up the microbiome.

Thus, in certain embodiments the present invention is directed todelivering to microbial cells in vivo a delivery vehicle with at leastone nucleic acid encoding a gene or nucleotide sequence of interest,such method employing an RNA-guided nuclease. The microbial cells may beeither or both pathogenic microbial cells or non-pathogenic bacterialcells and the gene or nucleotide sequence of interest may be a virulencefactor gene, a toxin gene, an antibiotic resistance gene, or amodulatory gene, and most preferably the nucleotide sequence of interestcomprises 16S ribosomal DNA (rDNA). Preferably the delivery vehicle is abacteriophage. After assessing what particular microbes are present in asample, the appropriate processing of such microbes using CRISPR-Cas todelete undesired genetic elements or features is relativelystraightforward (especially in view of the guidance provided herein andin conjunction with the references incorporated herein by reference.)

It has been observed by the present inventor that producing Haikuresembles the generation of a patent claim. There is requisitestructure, a need to communicate substance and

an ethereal quality of understanding. As one of skill in the art of bothbiology and Haiku will appreciate a cancer treatment that includes:

Selective killing

of your microbiome bugs

using CRISPR-Cas!

The foregoing has outlined rather broadly various pertinent andimportant features of various embodiments of the present invention. Suchdescription is, however, not to be considered as limiting the inventionin any way. The invention is capable of other embodiments and of beingpracticed and carried out in various ways which will become obvious tothose skilled in the art who read this specification. Also, it is to beunderstood that the phraseology and terminology employed herein are forthe purpose of description and should not be regarded as limiting of theinvention in any fashion.

While specific embodiments and applications of the present inventionhave been illustrated and described, it is to be understood that theinvention is not limited to the precise configuration and componentsdisclosed herein. Various modifications, changes, and variations whichwill be apparent to those skilled in the art may be made in thearrangement, operation, and details of the methods and systems of thepresent invention disclosed herein without departing from the spirit andscope of the invention. Those skilled in the art will appreciate thatthe conception upon which this disclosure is based, may readily beutilized as a basis for designing of other structures, methods andsystems for carrying out the several purposes of the present invention.It is important, therefore, that the claims be regarded as including anysuch equivalent construction insofar as they do not depart from thespirit and scope of the present invention.

1. A method for treating an individual suffering from a chronicinfectious disease and who has cancer, comprising, using a clusteredregularly interspaced short palindromic repeats (CRISPR) CRISPRassociated protein (Cas), selectively killing a pathogenic bacteriawithin the individual, said pathogenic bacteria selected from the groupconsisting of Staphylococcus aureus; Pseudomonas aeruginosa; Klebsiella;Streptoccocus; Salmonella; Shigella; Mycobacterium tuberculosis;Enterococcus; E coli; Clostridium; Neisseria gonnorrhoea; Acinetobacterbaumannii; and Campylobacter, and enhancing the growth of a beneficialbacteria in the individual selected from the group consisting ofAkkermansia, Bacteroides, Bifidobacterium, Clostridium, Enterococcus,Fusobacterium, Coprococcus, LactoBacillus, Propionibacterium,Ruminococcus, Veillonella, Prevotella, and Streptococcus bacteria;wherein said CRISPR Cas system comprises Cas3 and is delivered using abacteriophage.
 2. The method as set forth in claim 1, wherein the cancercomprises colorectal or bladder cancer.
 3. (canceled)
 4. The method asset forth in claim 1, further comprising administering to the individualan immune checkpoint inhibitor selected from the group consisting ofnivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110,TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012 andSTI-A1010.
 5. The method as set forth in claim 1, wherein using theCRISPR-Cas system, said pathogenic bacteria are killed while sparingother commensal bacteria.
 6. The method as set forth in claim 1, whereinthe pathogenic bacteria comprises Klebsiella pneumoniae.
 7. The methodas set forth in claim 1, wherein the CRISPR-Cas system is used to reducevirulence factors of the pathogenic bacteria.
 8. A method for treatingan individual suffering from a chronic infectious disease and who hascancer, comprising, using a clustered regularly interspaced shortpalindromic repeats (CRISPR) CRISPR associated protein (Cas) system or aCRISPR from Prevotella and Francisella 1 (Cpf1), selectively killingpathogenic bacteria within the individual, said pathogenic bacteriacomprising at least one of Staphylococcus aureus; Pseudomonasaeruginosa; Klebsiella; Streptoccocus; Salmonella; Shigella;Mycobacterium tuberculosis; Enterococcus; E coli; Clostridium; Neisseriagonnorrhoea; Acinetobacter baumannii; and Campylobacter; andadministering to the individual an immune checkpoint inhibitor thatspecifically binds to an immune checkpoint protein selected from thegroup consisting of CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA,KIR, LAG3, TIM-3 and VISTA.
 9. The method as set forth in claim 8,wherein the immune checkpoint inhibitor is selected from the groupconsisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514,STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C,AUR-012 and STI-A1010.
 10. The method as set forth in claim 8, whereinthe CRISPR-Cpf1 system is used to cut a gene expressed by the pathogenicbacteria.
 11. The method as set forth in claim 8, wherein the CRISPR-Casor Cpf1 system is used to insert genes that have controllable elementssuch that the pathogenic bacteria cells are killed by triggering theexpression of said inserted genes.
 12. The method as set forth in claim8, wherein the CRISPR-Cas system or Cpf1 system is delivered by abacteriophage.
 13. The method as set forth in claim 8, wherein saidpathogenic bacteria comprises at least one of Enterobacter aerogenes,Acinetobacter baumannii, and Klebsiella pneumoniae.
 14. The method ofclaim 8, further comprising enhancing the growth of a beneficialbacteria in the individual selected from the group consisting ofAkkermansia, Bacteroides, Bifidobacterium, Clostridium, Enterococcus,Fusobacterium, Coprococcus, LactoBacillus, Propionibacterium,Ruminococcus, Veillonella, Prevotella, and Streptococcus bacteria. 15.The method as set forth in claim 8, wherein the CRISPR-Cas or Cpf1system is used to cut a gene expressed by the pathogenic bacteria. 16.The method as set forth in claim 8, wherein CRISPR-Cas or Cpf1 is usedto insert antibacterial sensitivity into the genome of said pathogenicbacteria such that the pathogenic bacteria can selectively be killed.17. The method as set forth in claim 8, wherein CRISPR-Cas or Cpf1 isused to facilitate RNA-guided site-specific DNA cleavage to kill thepathogenic bacteria.
 18. The method as set forth in claim 8, whereinusing CRISPR-Cas systems, said pathogenic bacteria are killed whilesparing other commensal bacteria.
 19. The method as set forth in claim8, wherein using one of a CRISPR-Cas or Cpf1 system, said pathogenicbacteria are modified to reduce virulence factors of said pathogenicbacteria.
 20. (canceled)
 21. A method for treating an individualsuffering from a chronic infectious disease and cancer, comprising,using a clustered regularly interspaced short palindromic repeats(CRISPR) CRISPR associated protein (Cas) system from Prevotella andFrancisella 1 (Cpf1), selectively killing a pathogenic bacteria withinthe individual, said pathogenic bacteria selected from the groupconsisting of: Staphylococcus aureus; Pseudomonas aeruginosa;Klebsiella; Streptoccocus; Salmonella; Shigella; Mycobacteriumtuberculosis; Enterococcus; E coli; Clostridium; Neisseria gonnorrhoea;Acinetobacter baumannii; and Campylobacter; wherein the CRISPR-Cpf1system is used to cut a gene expressed by the pathogenic bacteria;wherein the CRISPR-Cpf1 system is used to facilitate RNA-guidedsite-specific DNA cleavage to kill the pathogenic bacteria; whereinusing the CRISPR-Cpf1 system said pathogenic bacteria are killed whilesparing other commensal bacteria; and wherein the CRISPR-Cpf1 system isdelivered to the pathogenic bacteria using a bacteriophage.
 22. Themethod as set forth in claim 21, wherein the CRISPR-Cpf1 system is usedto insert genes that have controllable elements such that the pathogenicbacteria cells are killed by triggering the expression of said insertedgenes.